N-acetylglucosaminyltransferase III expression in lower eukaryotes

ABSTRACT

The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII activity, which produce bisected N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/371,877, filed on Feb. 20, 2003, now issued U.S. Pat. No. 7,449,308,which is a continuation-in-part of U.S. application Ser. No. 09/892,591,filed Jun. 27, 2001, now issued U.S. Pat. No. 7,029,872, which claimsthe benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/214,358, filed Jun. 28, 2000, U.S. Provisional Application No.60/215,638, filed Jun. 30, 2000, and U.S. Provisional Application No.60/279,997, filed Mar. 30, 2001, each of which is incorporated herein byreference in its entirety. This application is also acontinuation-in-part of PCT/US02/41510, filed on Dec. 24, 2002, whichclaims the benefit of U.S. Provisional Application No. 60/344,169, filedon Dec. 27, 2001, each of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions by whichnon-human eukaryotic host cells, such as fungi or other eukaryoticcells, can be genetically modified to produce glycosylated proteins(glycoproteins) having patterns of glycosylation similar to those ofglycoproteins produced by animal cells, especially human cells, whichare useful as human or animal therapeutic agents.

BACKGROUND OF THE INVENTION Glycosylation Pathways in Humans and LowerEukaryotes

After DNA is transcribed and translated into a protein, furtherpost-translational processing involves the attachment of sugar residues,a process known as glycosylation. Different organisms produce differentglycosylation enzymes (glycosyltransferases and glycosidases), and havedifferent substrates (nucleotide sugars) available, so that theglycosylation patterns as well as composition of the individualoligosaccharides, even of the same protein, will be different dependingon the host system in which the particular protein is being expressed.Bacteria typically do not glycosylate proteins, and if so only in a veryunspecific manner (Moens and Vanderleyden (1997) Arch Microbiol.168(3):169-175). Lower eukaryotes such as filamentous fungi and yeastadd primarily mannose and mannosylphosphate sugars. The resulting glycanis known as a “high-mannose” type glycan or a mannan. Plant cells andinsect cells (such as Sf9 cells) glycosylate proteins in yet anotherway. By contrast, in higher eukaryotes such as humans, the nascentoligosaccharide side chain may be trimmed to remove several mannoseresidues and elongated with additional sugar residues that typically arenot found in the N-glycans of lower eukaryotes. See, e.g., Bretthauer,et al. (1999) Biotechnology and Applied Biochemistry 30:193-200;Martinet, et al. (1998) Biotechnology Letters 20:1171-1177; Weikert, etal. (1999) Nature Biotechnology, 17:1116-1121; M. Malissard, et al.(2000) Biochemical and Biophysical Research Communications 267:169-173;Jarvis, et al., (1998) Current Opinion in Biotechnology 9:528-533; andTakeuchi (1997) Trends in Glycoscience and Glycotechnology 9:S29-S35.

Synthesis of a mammalian-type oligosaccharide structure begins with aset of sequential reactions in the course of which sugar residues areadded and removed while the protein moves along the secretory pathway inthe host organism. The enzymes which reside along the glycosylationpathway of the host organism or cell determine the resultingglycosylation patterns of secreted proteins. Thus, the resultingglycosylation pattern of proteins expressed in lower eukaryotic hostcells differs substantially from the glycosylation pattern of proteinsexpressed in higher eukaryotes such as humans and other mammals(Bretthauer, 1999). The structure of a typical fungal N-glycan is shownin FIG. 1A.

The early steps of human glycosylation can be divided into at least twodifferent phases: (i) lipid-linked Glc₃Man₉GlcNAc₂ oligosaccharides areassembled by a sequential set of reactions at the membrane of theendoplasmic reticulum (ER) (FIG. 13) and (ii) the transfer of thisoligosaccharide from the lipid anchor dolichyl pyrophosphate onto denovo synthesized protein. The site of the specific transfer is definedby an asparagine (Asn) residue in the sequence Asn-Xaa-Ser/Thr where Xaacan be any amino acid except proline (Gavel and von Heijne (1990)Protein Eng. 3:433-42). Further processing by glucosidases andmannosidases occurs in the ER before the nascent glycoprotein istransferred to the early Golgi apparatus, where additional mannoseresidues are removed by Golgi specific alpha (α)-1,2-mannosidases.Processing continues as the protein proceeds through the Golgi. In themedial Golgi, a number of modifying enzymes, includingN-acetylglucosaminyl transferases (GnTI, GnTII, GnTIII, GnTIV and GnTV),mannosidase II and fucosyltransferases, add and remove specific sugarresidues. Finally, in the trans-Golgi, galactosyltranferases (GaIT) andsialyltransferases (ST) produce a glycoprotein structure that isreleased from the Golgi. It is this structure, characterized by bi-,tri- and tetra-antennary structures, containing galactose, fucose,N-acetylglucosamine and a high degree of terminal sialic acid, thatgives glycoproteins their human characteristics. The structure of atypical human N-glycan is shown in FIG. 1B. See also FIGS. 14 and 15 forsteps involved in mammalian-type N-glycan processing.

In nearly all eukaryotes, glycoproteins are derived from a commonlipid-linked oligosaccharide precursorGlc₃Man₉GlcNAc₂-dolichol-pyrophosphate. Within the endoplasmicreticulum, synthesis and processing of dolichol pyrophosphate boundoligosaccharides are identical between all known eukaryotes. However,further processing of the core oligosaccharide by fungal cells, e.g.,yeast, once it has been transferred to a peptide leaving the ER andentering the Golgi, differs significantly from humans as it moves alongthe secretory pathway and involves the addition of several mannosesugars.

In yeast, these steps are catalyzed by Golgi residingmannosyltransferases, like Och1p, Mnt1p and Mnn1p, which sequentiallyadd mannose sugars to the core oligosaccharide. The resulting structureis undesirable for the production of human-like proteins and it is thusdesirable to reduce or eliminate mannosyltransferase activity. Mutantsof S. cerevisiae, deficient in mannosyltransferase activity (for exampleoch1 or mnn9 mutants) have been shown to be non-lethal and displayreduced mannose content in the oligosaccharide of yeast glycoproteins.Other oligosaccharide processing enzymes, such as mannosylphosphatetransferase, may also have to be eliminated depending on the host'sparticular endogenous glycosylation pattern.

Sugar Nucleotide Precursors

The N-glycans of animal glycoproteins typically include galactose,fucose, and terminal sialic acid. These sugars are not found onglycoproteins produced in yeast and filamentous fungi. In humans, thefull range of nucleotide sugar precursors (e.g.,UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.) aresynthesized in the cytosol and transported into the Golgi, where theyare attached to the core oligosaccharide by glycosyltransferases.(Sommers and Hirschberg (1981) J. Cell Biol. 91(2):A406-A406; Sommersand Hirschberg (1982) J. Biol. Chem. 257(18):811-817; Perez andHirschberg (1987) Methods in Enzymology 138:709-715).

Glycosyl transfer reactions typically yield a side product which is anucleoside diphosphate or monophosphate. While monophosphates can bedirectly exported in exchange for nucleoside triphosphate sugars by anantiport mechanism, diphosphonucleosides (e.g., GDP) have to be cleavedby phosphatases (e.g. GDPase) to yield nucleoside monophosphates andinorganic phosphate prior to being exported. This reaction is importantfor efficient glycosylation; for example, GDPase from Saccharomycescerevisiae (S. cerevisiae) has been found to be necessary formannosylation. However that GDPase has 90% reduced activity toward UDP(Berninsone et al. (1994) J. Biol. Chem. 269(1):207-211). Lowereukaryotes typically lack UDP-specific diphosphatase activity in theGolgi since they do not utilize UDP-sugar precursors for Golgi-basedglycoprotein synthesis. Schizosaccharomyces pombe, a yeast found to addgalactose residues to cell wall polysaccharides (from UDP-galactose) hasbeen found to have specific UDPase activity, indicating the potentialrequirement for such an enzyme (Berninsone et al. (1994) J. Biol. Chem.269(1):207-211). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productmay be important to prevent glycosyl-transferase inhibition in the lumenof the Golgi (Khatara et al. (1974) Eur. J. Biochem. 44:537-560). SeeBerninsone et al. (1995) J. Biol. Chem. 270(24):14564-14567; Beaudet etal. (1998) Abc Transporters: Biochemical, Cellular, and MolecularAspects 292: 397-413.

Sequential Processing of N-Glycans by Compartmentalized EnzymeActivities

Sugar transferases and glycosidases (e.g., mannosidases) line the inner(luminal) surface of the ER and Golgi apparatus and thereby provide a“catalytic” surface that allows for the sequential processing ofglycoproteins as they proceed through the ER and Golgi network. Themultiple compartments of the cis, medial, and trans Golgi and thetrans-Golgi Network (TGN), provide the different localities in which theordered sequence of glycosylation reactions can take place. As aglycoprotein proceeds from synthesis in the ER to full maturation in thelate Golgi or TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmictail, individually or in concert, direct enzymes to the membrane ofindividual organelles and thereby localize the associated catalyticdomain to that locus (see, e.g., Gleeson (1998) Histochem. Cell Biol.109:517-532).

In some cases, these specific interactions were found to function acrossspecies. For example, the membrane spanning domain of α2,6-ST from rats,an enzyme known to localize in the trans-Golgi of the animal, was shownto also localize a reporter gene (invertase) in the yeast Golgi(Schwientek et al. (1995) J. Biol. Chem. 270(10):5483-9). However, thevery same membrane spanning domain as part of a full-length α2,6-ST wasretained in the ER and not further transported to the Golgi of yeast(Krezdorn et al. (1994) Eur. J. Biochem. 220(3):809-17). A full lengthGalT from humans was not even synthesized in yeast, despite demonstrablyhigh transcription levels. In contrast, the transmembrane region of thesame human GalT fused to an invertase reporter was able to directlocalization to the yeast Golgi, albeit it at low production levels.Schwientek and co-workers have shown that fusing 28 amino acids of ayeast mannosyltransferase (MNT1), a region containing a cytoplasmictail, a transmembrane region and eight amino acids of the stem region,to the catalytic domain of human GalT are sufficient for Golgilocalization of an active GalT. Other galactosyltransferases appear torely on interactions with enzymes resident in particular organellesbecause, after removal of their transmembrane region, they are stillable to localize properly.

Improper localization of a glycosylation enzyme may prevent properfunctioning of the enzyme in the pathway. For example, Aspergillusnidulans, which has numerous α-1,2-mannosidases (Eades and Hintz (2000)Gene 255(1):25-34), does not add GlcNAc to Man₅GlcNAc₂ when transformedwith the rabbit GnTI gene, despite a high overall level of GnTI activity(Kalsner et al. (1995) Glycoconj. J. 12(3):360-370). GnTI, althoughactively expressed, may be incorrectly localized such that the enzyme isnot in contact with both of its substrates: UDP-GlcNAc and a productiveMan₅GlcNAc₂ substrate (not all Man₅GlcNAc₂ structures are productive;see below). Alternatively, the host organism may not provide an adequatelevel of UDP-GlcNAc in the Golgi or the enzyme may be properly localizedbut nevertheless inactive in its new environment. In addition,Man₅GlcNAc₂ structures present in the host cell may differ in structurefrom Man₅GlcNAc₂ found in mammals. Maras and coworkers found that aboutone third of the N-glycans from cellobiohydrolase I (CBHI) obtained fromT. reesei can be trimmed to Man₅GlcNAc₂ by A. saitoi 1,2 mannosidase invitro. Fewer than 1% of those N-glycans, however, could serve as aproductive substrate for GnTI. Maras et al. (1997) Eur. J. Biochem.249:701-707. The mere presence of Man₅GlcNAc₂, therefore, does notassure that further in vivo processing of Man₅GlcNAc₂ can be achieved.It is formation of a productive, GnTI-reactive Man₅GlcNAc₂ structurethat is required. Although Man₅GlcNAc₂ could be produced in the cell(about 27 mol %), only a small fraction could be converted toMan₅GlcNAc₂ (less than about 5%, see Chiba et al. WO 01/14522).

To date, there is no reliable way of predicting whether a particularheterologously expressed glycosyltransferase or mannosidase in a lowereukaryote will be (1), sufficiently translated (2), catalytically activeor (3) located to the proper organelle within the secretory pathway.Because all three of these are necessary to affect glycosylationpatterns in lower eukaryotes, a systematic scheme to achieve the desiredcatalytic function and proper retention of enzymes in the absence ofpredictive tools, which are currently not available, would be desirable.

Production of Therapeutic Glycoproteins

A significant number of proteins isolated from humans or animals arepost-translationally modified, with glycosylation being one of the mostsignificant modifications. An estimated 70% of all therapeutic proteinsare glycosylated and thus currently rely on a production system (i.e.,host cell) that is able to glycosylate in a manner similar to humans.Several studies have shown that glycosylation plays an important role indetermining the (1) immunogenicity, (2) pharmacokinetic properties, (3)trafficking, and (4) efficacy of therapeutic proteins. It is thus notsurprising that substantial efforts by the pharmaceutical industry havebeen directed at developing processes to obtain glycoproteins that areas “humanoid” or “human-like” as possible. To date, most glycoproteinsare made in a mammalian host system. This may involve the geneticengineering of such mammalian cells to enhance the degree of sialylation(i.e., terminal addition of sialic acid) of proteins expressed by thecells, which is known to improve pharmacokinetic properties of suchproteins. Alternatively, one may improve the degree of sialylation by invitro addition of such sugars using known glycosyltransferases and theirrespective nucleotide sugars (e.g., 2,3-sialyltransferase and CMP-sialicacid).

While most higher eukaryotes carry out glycosylation reactions that aresimilar to those found in humans, recombinant human proteins expressedin the above mentioned host systems invariably differ from their“natural” human counterpart (Raju et al. (2000) Glycobiology 10(5):477-486). Extensive development work has thus been directed at findingways to improve the “human character” of proteins made in theseexpression systems. This includes the optimization of fermentationconditions and the genetic modification of protein expression hosts byintroducing genes encoding enzymes involved in the formation ofhuman-like glycoforms. Goochee et al. (1999) Biotechnology9(12):1347-55; Andersen and Goochee (1994) Curr Opin Biotechnol.5(5):546-49; Werner et al. (1998) Arzneimittelforschung. 48(8):870-80;Weikert et al. (1999) Nat Biotechnol. 17(11):1116-21; Yang and Butler(2000) Biotech. Bioeng. 68:370-80. Inherent problems associated with allmammalian expression systems have not been solved.

Glycoprotein Production Using Eukaryotic Microorganisms

Although the core oligosaccharide structure transferred to a protein inthe endoplasmic reticulum is basically identical in mammals and lowereukaryotes, substantial differences have been found in the subsequentprocessing reactions which occur in the Golgi apparatus of fungi andmammals. In fact, even amongst different lower eukaryotes there exist agreat variety of glycosylation structures. This has historicallyprevented the use of lower eukaryotes as hosts for the production ofrecombinant human glycoproteins despite otherwise notable advantagesover mammalian expression systems.

Therapeutic glycoproteins produced in a microorganism host such as yeastutilizing the endogenous host glycosylation pathway differ structurallyfrom those produced in mammalian cells and typically show greatlyreduced therapeutic efficacy. Such glycoproteins are typicallyimmunogenic in humans and show a reduced half-life (and thusbioactivity) in vivo after administration (Takeuchi (1997) Trends inGlycoscience and Glycotechnology 9:S29-S35). Specific receptors inhumans and animals (i.e., macrophage mannose receptors) can recognizeterminal mannose residues and promote the rapid clearance of the foreignglycoprotein from the bloodstream. Additional adverse effects mayinclude changes in protein folding, solubility, susceptibility toproteases, trafficking, transport, compartmentalization, secretion,recognition by other proteins or factors, antigenicity, orallergenicity.

Yeast and filamentous fungi have both been successfully used for theproduction of recombinant proteins, both intracellular and secreted(Cereghino and Cregg (2000) FEMS Microbiology Reviews 24(1):45-66;Harkki et al. (1989) Bio-Technology 7(6):596; Berka et al. (1992) Abstr.Papers Amer. Chem. Soc. 203:121-BIOT; Svetina et al. (2000) J.Biotechnol. 76(2-3):245-251). Various yeasts, such as K. lactis, Pichiapastoris, Pichia methanolica, and Hansenula polymorpha, have playedparticularly important roles as eukaryotic expression systems becausethey are able to grow to high cell densities and secrete largequantities of recombinant protein. Likewise, filamentous fungi, such asAspergillus niger, Fusarium sp, Neurospora crassa and others, have beenused to efficiently produce glycoproteins at the industrial scale.However, as noted above, glycoproteins expressed in any of theseeukaryotic microorganisms differ substantially in N-glycan structurefrom those in animals. This has prevented the use of yeast orfilamentous fungi as hosts for the production of many therapeuticglycoproteins.

Although glycosylation in yeast and fungi is very different than inhumans, some common elements are shared. The first step, the transfer ofthe core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g., OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyltransferase activity. Mutants of S. cerevisiaedeficient in mannosyltransferase activity (e.g., och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosaccharide of yeast glycoproteins. Other oligosaccharide processingenzymes, such as mannosylphosphate transferase, may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions,the formation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to localize theseenzymes so that a sequential processing of the maturing glycosylationstructure is ensured.

Several efforts have been made to modify the glycosylation pathways ofeukaryotic microorganisms to provide glycoproteins more suitable for useas mammalian therapeutic agents. For example, severalglycosyltransferases have been separately cloned and expressed in S.cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and other fungi(Yoshida et al. (1999) Glycobiology 9(1):53-8, Kalsner et al. (1995)Glycoconj. J 12(3):360-370). However, N-glycans resembling those made inhuman cells were not obtained.

Yeasts produce a variety of mannosyltransferases (e.g.,1,3-mannosyltransferases such as MNN1 in S. cerevisiae; Graham and Emr(1991) J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g.,KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (e.g., OCH1from S. cerevisiae), mannosylphosphate transferases and their regulators(e.g., MNN4 and MNN6 from S. cerevisiae) and additional enzymes that areinvolved in endogenous glycosylation reactions. Many of these genes havebeen deleted individually giving rise to viable organisms having alteredglycosylation profiles. Examples are shown in Table 1.

TABLE 1 Examples of yeast strains having altered mannosylation StrainN-glycan (wild type) Mutation N-glycan (mutant) Reference S. pombeMan_(>9)GlcNAc₂ OCH1 Man₈GlcNAc₂ Yoko-o et al. (2001) FEBS Lett. 489(1):75-80 S. cerevisiae Man_(>9)GlcNAc₂ OCH1/MNN1 Man₈GlcNAc₂Nakanishi-Shindo et al. (1993) J. Biol. Chem. 268(35): 26338-26345 S.cerevisiae Man_(>9)GlcNAc₂ OCH1/MNN1/MNN4 Man₈GlcNAc₂ Chiba et al.(1998) J. Biol. Chem. 273, 26298-26304 P. pastoris HyperglycosylatedOCH1 (complete Not Welfide, Japanese deletion) hyperglycosylatedApplication Publication No. 8- 336387 P. pastoris Man_(>8)GlcNAc₂ OCH1(disruption) Man_(>8)GlcNAc₂ Contreras et al. WO 02/00856 A2

Japanese Patent Application Publication No. 8-336387 discloses thedeletion of an OCH1 homolog in Pichia pastoris. In S. cerevisiae, OCH1encodes a 1,6-mannosyltransferase, which adds a mannose to the glycanstructure Man₈GlcNAc₂ to yield Man₉GlcNAc₂. The Man₉GlcNAc₂ structure,which contains three 1,6 mannose residues, is then a substrate forfurther 1,2-, 1,6-, and 1,3-mannosyltransferases in vivo, leading to thehypermannosylated glycoproteins that are characteristic for S.cerevisiae and which typically may have 30-40 mannose residues perN-glycan. Because the Och1p initiates the transfer of 1,6 mannose to theMan₈GlcNAc₂ core, it is often referred to as the “initiating 1,6mannosyltransferase” to distinguish it from other 1,6mannosyltransferases acting later in the Golgi. In an och1 mnn1 mnn4mutant strain of S. cerevisiae, proteins glycosylated with Man₈GlcNAc₂accumulate and hypermannosylation does not occur. However, Man₈GlcNAc₂is not a substrate for mammalian glycosyltransferases, such as humanUDP-GlcNAc transferase I, and accordingly, the use of that mutantstrain, in itself, is not useful for producing mammalian-like proteins,i.e., those with complex or hybrid glycosylation patterns.

One can trim Man₈GlcNAc₂ structures to a Man₅GlcNAc₂ isomer in S.cerevisiae (although high efficiency trimming greater than 50% in vivohas yet to be demonstrated) by engineering a fungal mannosidase from A.saitoi into the endoplasmic reticulum (ER). The shortcomings of thisapproach are two-fold: (1) it is not clear whether the Man₅GlcNAc₂structures formed are in fact formed in vivo (rather than having beensecreted and further modified by mannosidases outside the cell); and (2)it is not clear whether any Man₅GlcNAc₂ structures formed, if in factformed in vivo, are the correct isoform to be a productive substrate forsubsequent N-glycan modification by GlcNAc transferase I (Maras et al.(1997) Eur. J Biochem. 249:701-707).

With the objective of providing a more human-like glycoprotein derivedfrom a fungal host, U.S. Pat. No. 5,834,251 discloses a method forproducing a hybrid glycoprotein derived from Trichoderma reseei. Ahybrid N-glycan has only mannose residues on the Manα1-6 arm of the coremannose structure and one or two complex antennae on the Manα1-3 arm.While this structure has utility, the method has the disadvantage thatnumerous enzymatic steps must be performed in vitro, which is costly andtime-consuming. Isolated enzymes are expensive to prepare and needcostly substrates (e.g., UDP-GlcNAc). The method also does not allow forthe production of complex glycans on a desired protein.

Intracellular Mannosidase Activity Involved in N-Glycan Trimming

Alpha-1,2-mannosidase activity is required for the trimming ofMan₈GlcNAc₂ to form Man₅GlcNAc₂, which is a major intermediate forcomplex N-glycan formation in mammals. Previous work has shown thattruncated murine, fungal and human α-1,2-mannosidase can be expressed inthe methylotropic yeast P. pastoris and display Man₈GlcNAc₂ toMan₅GlcNAc₂ trimming activity (Lal et al. (1998) Glycobiology8(10):981-95; Tremblay et al. (1998) Glycobiology 8(6):585-95,Callewaert et al. (2001) FEBS Lett. 503(2-3):173-8). However, to date,no reports exist that show the high level in vivo trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ on a secreted glycoprotein from P. pastoris.

Moreover, the mere presence of an α-1,2-mannosidase in the cell doesnot, by itself, ensure proper intracellular trimming of Man₈GlcNAc₂ toMan₅GlcNAc₂. (See, e.g., Contreras et al. WO 02/00856 A2, in which anHDEL tagged mannosidase of T. reesei is localized primarily in the ERand co-expressed with an influenza haemagglutinin (HA) reporter proteinon which virtually no Man₅GlcNAc₂ could be detected. See also Chiba etal. (1998) J. Biol. Chem. 273(41): 26298-26304, in which a chimericα-1,2-mannosidase/Och1p transmembrane domain fusion localized in the ER,early Golgi and cytosol of S. cerevisiae, had no mannosidase trimmingactivity). Accordingly, mere localization of a mannosidase in the ER orGolgi is insufficient to ensure activity of the respective enzyme inthat targeted organelle. (See also, Martinet et al. (1998) Biotech.Letters 20(12): 1171-1177, showing that α-1,2-mannosidase from T.reesei, while localizing intracellularly, increased rather thandecreased the extent of mannosylation). To date, there is no report thatdemonstrates the intracellular localization of an active heterologousα-1,2-mannosidase in either yeast or fungi using a transmembranelocalization sequence.

While it is useful to engineer strains that are able to produceMan₅GlcNAc₂ as the primary N-glycan structure, any attempt to furthermodify these high mannose precursor structures to more closely resemblehuman glycans requires additional in vivo or in vitro steps. Methods tofurther humanize glycans from fungal and yeast sources in vitro aredescribed in U.S. Pat. No. 5,834,251 (supra). If Man₅GlcNAc₂ is to befurther humanized in vivo, one has to ensure that the generatedMan₅GlcNAc₂ structures are, in fact, generated intracellularly and notthe product of mannosidase activity in the medium. Complex N-glycanformation in yeast or fungi will require high levels of Man₅GlcNAc₂ tobe generated within the cell because only intracellular Man₅GlcNAc₂glycans can be further processed to hybrid and complex N-glycans invivo. In addition, one has to demonstrate that the majority ofMan₅GlcNAc₂ structures generated are in fact a substrate for GnTI andthus allow the formation of hybrid and complex N-glycans.

Accordingly, the need exists for methods to produce glycoproteinscharacterized by a high intracellular Man₅GlcNAc₂ content which can befurther processed into human-like glycoprotein structures in non-humaneukaryotic host cells, and particularly in yeast and filamentous fungi.

N-Acetylglucosaminyltransferases

N-Acetylglucosaminyltransferases (“GnTs”) belong to another class ofglycosylation enzymes that modify N-linked oligosaccharides in thesecretory pathway. Such glycosyltransferases catalyze the transfer of amonosaccharide from specific sugar nucleotide donors onto particularhydroxyl position of a monosaccharide in a growing glycan chain in oneof two possible anomeric linkages (either α or β). Dennis et al. (1999)Bioessays 21(5):412-21. Specific GnTs add N-acetylglucosamine (“GlcNAc”)onto the Manα1,6 arm or the Manα1,3 arm of an N-glycan substrate (e.g.,Man₅GlcNAc₂ (“mannose-5 core”) and Man₃GlcNAc₂ (an “inner corestructure”)). The reaction product (e.g., GlcNAcMan₅GlcNAc₂ orGlcNAc₂Man₃GlcNAc₂) can then be modified into bi-, tri-, andtetra-antennary N-linked oligosaccharide structures.

N-Acetylglucosaminyltransferase III (“GnTIII”) is an enzyme thatcatalyzes the addition of a GlcNAc, on the middle mannose of thetrimannose core (Manα1,6 (Manα1,3) Man β1,4-GlcNAc β1,4-GlcNAc β1,4-Asn)of an N-linked oligosaccharide. The addition by GnTIII of a bisectingGlcNAc to an acceptor substrate (e.g. trimannose core) yields aso-called bisected N-glycan. For example, the addition by GnTIII of abisecting GlcNAc to the GlcNAcMan₃GlcNAc₂ structure may yield a bisectedN-glycan, GlcNAc₂Man₃GlcNAc₂. Similarly, the addition by GnTIII of abisecting GlcNAc to a GlcNAc₂Man₃GlcNAc₂ structure yields anotherbisected N-glycan, GlcNAc₃Man₃GlcNAc₂. This latter structure has beenimplicated in greater antibody-dependent cellular cytotoxicity (ADCC).Umana et al. (1999) Nat. Biotechnol. 17(2): 176-80. Other bisectedN-glycans can be formed by the action of GnTIII. For example,GlcNAcMan₄GlcNAc₂ can be converted to bisected GlcNAc₂Man₄GlcNAc₂,Man₅GlcNAc₂ can be converted to bisected GlcNAcMan₅GlcNAc₂, andGlcNAcMan₅GlcNAc₂ can be converted to bisected GlcNAc₂Man₅GlcNAc₂. See,e.g., Narasimhan (1982) J. Biol. Chem. 257:10235-42. Thus far, GnTIIIactivity has only been shown in mammalian cells.

Re-engineering glycoforms of immunoglobulins expressed by mammaliancells is a tedious and cumbersome task. Especially in the case ofGnTIII, where over-expression of this enzyme has been implicated ingrowth inhibition, methods involving regulated (inducible) geneexpression had to be employed to produce immunoglobulins with bisectedN-glycans. Umana et al. (1999) Biotechnol Bioeng. 65(5):542-9; Umana etal. (1999) Nat. Biotechnol. 17(2):176-80; Umana et al WO 03/011878; U.S.Pat. No. 6,602,684. Such a growth-inhibition effect complicates theability to coexpress the target protein and GnTIII and may impose anupper limit on GnTIII overexpression. U.S. Pat. No. 6,602,684. Carefuloptimization of the expression levels of GnTIII may be necessary. Id.What is needed, therefore, is a protein production system utilizing theinherent capability of robust product titers such as those produced inlower eukaryotic host cells (e.g., yeast and filamentous fungi), whichis capable of producing bisected N-glycans on proteins, especiallytherapeutic proteins, expressed in these cells. As described above,however, development of the lower eukaryotic host cells used in such aprotein production system requires that the endogenous glycosylationpathways of the host cells be further modified.

SUMMARY OF THE INVENTION

Host cells and cell lines having genetically modified glycosylationpathways that allow them to carry out a sequence of enzymatic reactionswhich mimic the processing of glycoproteins in mammals, especially inhumans, have been developed. Recombinant proteins expressed in theseengineered hosts yield glycoproteins more similar, if not substantiallyidentical, to their mammalian, e.g., human counterparts. Host cells ofthe invention, e.g., lower eukaryotic microorganisms and othernon-human, eukaryotic host cells grown in culture, are modified toproduce N-glycans, such as bisected N-glycans, or other structuresproduced along human glycosylation pathways. This result is achievedusing a combination of engineering and/or selection of strains that donot, for example, express enzymes that create the undesirable structurescharacteristic of the fungal glycoproteins and that do, for example,express heterologous enzymes capable of producing a “human-like”glycoprotein.

The present invention thus provides a glycoprotein production methodusing (1) a lower eukaryotic host such as a unicellular or filamentousfungus, or (2) any non-human eukaryotic organism that has a differentglycosylation pattern from humans, to modify the glycosylationcomposition and structures of the proteins made in a host organism(“host cell”) so that they resemble more closely carbohydrate structuresfound in mammalian, e.g., human proteins. The process allows one toobtain an engineered host cell which can be used to express and targetany desirable gene(s), e.g., one involved in glycosylation, by methodsthat are well-established in the scientific literature and generallyknown to the artisan in the field of protein expression. Host cells withmodified oligosaccharides are created or selected. For the production oftherapeutic proteins, this method may be adapted to engineer cell linesin which any desired glycosylation structure may be obtained.

Accordingly, in one embodiment, the invention provides methods formaking a human-like glycoprotein in a lower eukaryotic host cell byintroduction into the cell of an N-acetylglucosaminyltransferase IIIactivity. In a preferred embodiment, the N-acetylglucosaminyltransferaseIII activity is expressed in the cell, and in an even more preferredembodiment, this expression results in the production of N-glycanscomprising GlcNAc₃Man₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, or GlcNAc₂Man₅GlcNAc₂bisected structures. In another preferred embodiment, theN-acetylglucosaminyltransferase III activity is substantiallyintracellular. In another preferred embodiment of the invention, theglycoprotein including the N-glycans with bisected structures isisolated from the lower eukaryotic host cell. In an even more preferredembodiment, the glycoprotein produced in the host cell is a therapeuticprotein.

In another aspect, the invention provides a lower eukaryotic host cellthat includes both an N-acetylglucosaminyltransferase III activity andan N-acetylglucosaminyltransferase II activity. In a preferredembodiment, the host cell including the N-acetylglucosaminyltransferaseIII activity produces N-glycans comprising GlcNAcMan₃GlcNAc₂ structuresthat are capable of reacting with this activity. In a more preferredembodiment, the activity produces a bisected glycan. The lowereukaryotic host cell of some embodiments of the invention may thusinclude an N-glycan with a bisected glycan. In a preferred embodiment,the N-glycan includes greater than 10 mole % of the bisected glycan. Insome embodiments, the host cell includes an N-glycan that comprisesGlcNAc₃Man₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, or GlcNAc₂Man₅GlcNAc₂ bisectedstructures. In a preferred embodiment, the host cell includes aMan₅GlcNAc₂ core structure or a Man₃GlcNAc₂ core structure that ismodified by a bisecting GlcNAc. In an even more preferred embodiment,the cell produces greater than 10 mole % of the modified structure.

In another embodiment of the invention, the lower eukaryotic host cellcontains an N-acetylglucosaminyltransferase I activity in addition tothe N-acetylglucosaminyltransferase III activity. In a preferredembodiment, the activities are substantially intracellular. In anotherpreferred embodiment, the cell produces N-glycans comprisingGlcNAcMan₃GlcNAc₂ that are capable of reacting with the GnTIII activity.In an even more preferred embodiment, the GnTIII activity of the cellproduces a bisected glycan.

In another embodiment, the lower eukaryotic host cell of the inventioncontains both an N-acetylglucosaminyltransferase III activity and amannosidase II activity. In a preferred embodiment, the host cellfurther contains an N-acetylglucosaminyltransferase I activity. Inanother preferred embodiment, the host cell further contains anN-acetylglucosaminyltransferase II activity. In another preferredembodiment, the host cell further contains both anN-acetylglucosaminyltransferase I activity and anN-acetylglucosaminyltransferase II activity.

In another embodiment, the host cell of the invention is deficient in anOCH1 mannosyltransferase activity. Such a cell may, for example, bedeficient in a Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferaseactivity. In yet another embodiment, the host cell of the invention mayfurther comprise an α-1,2-mannosidase I activity. In another embodiment,the host cell may further comprise a UDP-GlcNAc transporter.

The present invention also provides glycoproteins that are made by theprocesses of the invention. In one embodiment, the glycoprotein includesa bisecting GlcNAc on a Man₅GlcNAc₂ or a Man₃GlcNAc₂ core structure andis produced in a lower eukaryotic host cell. In another embodiment, theglycoprotein includes a bisecting GlcNAc attached to a Man₅GlcNAc₂,Man₄GlcNAc₂. Man₃GlcNAc₂, GlcNAcMan₃GlcNAc₂, GlcNAcMan₅GlcNAc₂, or aGlcNAc₂Man₃GlcNAc₂ core structure and is produced in a lower eukaryotichost cell. In a preferred embodiment, greater than 10 mole % of the corestructures of the glycoprotein of the invention are modified by thebisecting GlcNAc.

In another aspect, the invention provides pharmaceutical compositionsthat contain the human-like glycoproteins produced in a lower eukaryotichost cell. Also provided according to the invention are vectors encodingproteins having N-acetylglucosaminyltransferase III activity andcontaining attached targeting peptide sequences. In a preferredembodiment, the proteins encoded by the vectors are localized in a lowereukaryotic host cell such that they produce N-glycans having bisectedstructures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a typical fungal N-glycosylationpathway.

FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

FIG. 2 depicts construction of a combinatorial DNA library of fusionconstructs. FIG. 2A diagrams the insertion of a targeting peptidefragment into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.). FIG. 2B showsthe generated targeting peptide sub-library having restriction sitesNotI-AscI. FIG. 2C diagrams the insertion of a catalytic domain regioninto pJN347, a modified pUC19 vector. FIG. 2D shows the generatedcatalytic domain sub-library having restriction sites NotI, AscI andPacI. FIG. 2E depicts one particular fusion construct generated from thetargeting peptide sub-library and the catalytic domain sub-library.

FIG. 3 illustrates the M. musculus α-1,2-mannosidase IA open readingframe nucleic acid sequence (SEQ ID NO:50) and encoded polypeptidesequence (SEQ ID NO:51). The sequences of the PCR primers used togenerate N-terminal truncations are underlined.

FIG. 4 illustrates engineering of vectors with multiple auxotrophicmarkers and genetic integration of target proteins in the P. pastorisOCH1 locus.

FIGS. 5A-5E show MALDI-TOF analysis demonstrating production of kringle3 domain of human plasminogen (K3) glycoproteins having Man₅GlcNAc₂ asthe predominant N-glycan structure in P. pastoris. FIG. 5A depicts thestandard Man₅GlcNAc₂ [a] glycan (Glyko, Novato, Calif.) andMan₅GlcNAc₂+Na⁺[b]. FIG. 5B shows PNGase-released glycans from K3 wildtype. The N-glycans shown are as follows: Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]. FIG. 5C depicts the och1deletion resulting in the production of Man₈GlcNAc₂ [c] as thepredominant N-glycan. FIGS. 5D and 5E show the production of Man₅GlcNAc₂[b] after in vivo trimming of Man₈GlcNAc₂ with a chimericα-1,2-mannosidase. The predominant N-glycan is indicated by a peak witha mass (m/z) of 1253 consistent with its identification as Man₅GlcNAc₂[b].

FIGS. 6A-6F show MALDI-TOF analysis demonstrating production of IFN-βglycoproteins having Man₅GlcNAc₂ as the predominant N-glycan structurein P. pastoris. FIG. 6A shows the standard Man₅GlcNAc₂ [a] andMan₅GlcNAc₂+Na⁺ [b] as the standard (Glyko, Novato, Calif.). FIG. 6Bshows PNGase-released glycans from IFN-β wildtype. FIG. 6C depicts theoch1 knock-out producing Man₈GlcNAc₂ [c]; Man₉GlcNAc₂ [d]; Man₁₀GlcNAc₂[e]; Man₁₁GlcNAc₂ [f]; Man₁₂GlcNAc₂ [g]; and no production ofMan₅GlcNAc₂ [b]. FIG. 6D shows relatively small amount of Man₅GlcNAc₂[b] among other intermediate N-glycans Man₈GlcNAc₂ [c] to Man₁₂GlcNAc₂[g]. FIG. 6E shows a significant amount of Man₅GlcNAc₂ [b] relative tothe other glycans Man₈GlcNAc₂ [c] and Man₉GlcNAc₂ [d] produced by pGC5(Saccharomyces MNS1(m)/mouse mannosidase IB Δ99). FIG. 6F showspredominant production of Man₅GlcNAc₂ [b] on the secreted glycoproteinIFN-β by pFB8 (Saccharomyces SEC12(m)/mouse mannosidase IA Δ187). TheN-glycan is indicated by a peak with a mass (m/z) of 1254 consistentwith its identification as Man₅GlcNAc₂ [b].

FIG. 7 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pFB8 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 8 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pGC5 mannosidase, which demonstratesa lack of extracellular mannosidase activity in the supernatant; and (C)Man₉GlcNAc₂ standard labeled with 2-AB after exposure to T. reeseimannosidase (positive control).

FIG. 9 shows a high performance liquid chromatogram for: (A) Man₉GlcNAc₂standard labeled with 2-AB (negative control); (B) supernatant of mediumP. pastoris, Δoch1 transformed with pBC18-5 mannosidase, whichdemonstrates lack of extracellular mannosidase activity in thesupernatant; and (C) supernatant of medium P. pastoris, Δoch1transformed with pDD28-3, which demonstrates activity in the supernatant(positive control).

FIGS. 10A-10B demonstrate the activity of an UDP-GlcNAc transporter inthe production of GlcNAcMan₅GlcNAc₂ in P. pastoris. FIG. 10A depicts aP. pastoris strain (YSH-3) with a human GnTI but without the UDP-GlcNActransporter resulting in some production of GlcNAcMan₅GlcNAc₂ [b] but apredominant production of Man₅GlcNAc₂ [a]. FIG. 10B depicts the additionof UDP-GlcNAc transporter from K. lactis in a strain (PBP-3) with thehuman GnTI, which resulted in the predominant production ofGlcNAcMan₅GlcNAc₂ [b]. The single prominent peak of mass (m/z) at 1457is consistent with its identification as GlcNAcMan₅GlcNAc₂ [b] as shownin FIG. 10B.

FIG. 11 shows a pH optimum of a heterologous mannosidase enzyme encodedby pBB27-2 (Saccharomyces MNN10(s)/C. elegans mannosidase IB Δ31)expressed in P. pastoris.

FIGS. 12A-12C show MALDI-TOF analysis of N-glycans released from a cellfree extract of K. lactis. FIG. 12A shows the N-glycans released fromwild-type cells, which includes high-mannose type N-glycans. FIG. 12Bshows the N-glycans released from och1 mnn1 deleted cells, revealing adistinct peak of mass (m/z) at 1908 consistent with its identificationas Man₉GlcNAc₂ [d]. FIG. 12C shows the N-glycans released from och1 mnn1deleted cells after in vitro α-1,2-mannosidase digest corresponding to apeak consistent with Man₅GlcNAc₂.

FIG. 13 is a schematic of the structure of the dolichylpyrophosphate-linked oligosaccharide.

FIG. 14 is a schematic of the generation of GlcNAc₂Man₃GlcNAc₂ N-glycansfrom fungal host cells which are deficient in alg3, alg9, or alg12activities.

FIG. 15 is a schematic of processing reactions required to producemammalian-type oligosaccharide structures in a fungal host cell with analg3, och1 genotype.

FIG. 16 shows S. cerevisiae Alg3 Sequence Comparisons (Blast) (SEQ IDNOs:9-20, respectively, in order of appearance)

FIG. 17 shows S. cerevisiae ALG3 (SEQ ID NO:21) and Alg3p (SEQ ID NO:22)Sequences

FIG. 18 shows P. pastoris ALG3 (SEQ ID NO:23) and Alg3p (SEQ ID NO:24)Sequences

FIG. 19 shows P. pastoris ALG3 Sequence Comparisons (Blast) (SEQ IDNO:25-32, respectively, in order of appearance)

FIG. 20 shows K. lactis ALG3 (SEQ ID NO:33) and Alg3p (SEQ ID NO:34)Sequences

FIG. 21 shows K. lactis ALG3 Sequence Comparisons (Blast) (SEQ IDNOs:35-40, respectively, in order of appearance)

FIG. 22 shows a model of an IgG immunoglobulin. Heavy chain and lightchain can be, based on similar secondary and tertiary structure,subdivided into domains. The two heavy chains (domains V_(H), C_(H)1,C_(H)2 and C_(H)3) are linked through three disulfide bridges. The lightchains (domains V_(L) and C_(L)) are linked by another disulfide bridgeto the C_(H)1 portion of the heavy chain and, together with the C_(H)1and V_(H) fragments, make up the Fab region. Antigens bind to theterminal portion of the Fab region. Effector-functions, such asFc-gamma-Receptor binding have been localized to the C_(H)2 domain, justdownstream of the hinge region and are influenced by N-glycosylation ofasparagine 297 in the heavy chain.

FIG. 23 is a schematic overview of a modular IgG1 expression vector.

FIG. 24 shows M. musculis GnTIII Nucleic Acid (SEQ ID NO:45) And AminoAcid (SEQ ID NO:46) Sequences

FIG. 25 (top) is a MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in a P. pastoris YSH-1 displaying apredominant peak at 1461 m/z corresponding to the the mass ofGlcNAcMan₅GlcNAc₂ [d]; FIG. 25 (bottom) shows a MALDI-TOF-MS analysis ofN-glycans isolated from a kringle 3 glycoprotein produced in a P.pastoris YSH-1 transformed with D. melanogaster mannosidase IIΔ74/S.cerevisiae MNN2(s) showing a predominant peak at 1140 m/z correspondingto the mass of GlcNAcMan₃GlcNAc₂ [b] and other peaks corresponding toGlcNAcMan₄GlcNAc₂ [c] at 1303 m/z and GlcNAcMan₅GlcNAc₂ [d] at 1465 m/z.This strain was designated YSH-37.

FIG. 26 (top) is the MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH-1 as shown in FIG. 25(top); FIG. 26 (bottom) is a MALDI-TOF-MS analysis of N-glycans isolatedfrom a kringle 3 glycoprotein expressed in P. pastoris YSH-1 cellstransformed with a pVA53 construct (S. cerevisiae MNN2(s)/mGnTIII). Thepeak at 1463 m/z corresponds the mass of GlcNAcMan₅GlcNAc₂ [d] and thepeak at 1666 m/z corresponds to the mass of GlcNAc₂Man₅GlcNAc₂ [a].

FIG. 27 (top) is the MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH-1 as shown in FIG. 25(top); FIG. 27 (bottom) is a MALDI-TOF-MS analysis of N-glycans isolatedfrom a kringle 3 glycoprotein expressed in P. pastoris YSH-1 cellstransformed with a pVA55 construct (S. cerevisiae MNN2(s)/mGnTIII). Thepeak at 1463 m/z corresponds to the mass of GlcNAcMan₅GlcNAc₂ [d] andthe peak at 1667 m/z corresponds to the mass of GlcNAc₂Man₅GlcNAc₂ [a].

FIG. 28 (top) is the MALDI-TOF-MS analysis of N-glycans isolated from akringle 3 glycoprotein produced in P. pastoris YSH-1 as shown in FIG. 25(top); FIG. 28 (bottom) is a MALDI-TOF-MS analysis of N-glycans isolatedfrom a kringle 3 glycoprotein expressed in P. pastoris YSH-1 cellstransformed with a pVB51 construct (K. lactis GNT1(s)/mGnTIII). Thepredominant peak at 1463 m/z corresponds to the mass ofGlcNAcMan₅GlcNAc₂ [d] and a second peak at 1726 m/z [e], which does notcorrepond to the mass of GlcNAc₂Man₅GlcNAc₂ is observed.

FIG. 29 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle3 glycoprotein expressed in P. pastoris YSH-44 cells. The predominantpeak at 1356 m/z corresponds to the mass of GlcNAc₂Man₃GlcNAc₂ [x].

FIG. 30 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle3 glycoprotein expressed in P. pastoris YSH-44 cells transformed with apVA53 construct (S. cerevisiae MNN2(s)/mGnTIII). The peak at 1340 m/zcorresponds to the mass of GlcNAc₂Man₃GlcNAc₂ [x] and the peak at 1542m/z corresponds to the mass of GlcNAc₃Man₃GlcNAc₂ [y].

FIG. 31 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle3 glycoprotein expressed in P. pastoris PBP6-5 cells. The predominantpeak at 1340 m/z corresponds to the mass of GlcNAc₂Man₃GlcNAc₂ [x].

FIG. 32 is a MALDI-TOF-MS analysis of N-glycans isolated from a kringle3 glycoprotein expressed in P. pastoris PBP6-5 cells transformed with apVA53 construct (S. cerevisiae MNN2(s)/mGnTIII). The peak at 1340 m/zcorresponds to the mass of GlcNAc₂Man₃GlcNAc₂ [x] and the peak at 1543m/z corresponds to the mass of GlcNAc₃Man₃GlcNAc₂ [y].

FIG. 33 shows a high performance liquid chromatogram, which demonstratesa lack of extracellular GnTIII activity (pVA53) in the supernatant. TheN-glycan GlcNAcMan₅GlcNAc₂ purified from K3 expressed in PBP-3 strainwas added to: BMMY (A); 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis,Mo.)) in BMMY (B); the supernatant of YSH-44 transformed with pVA53[YSH-57] (C); and the supernatant of YSH-57+1 mM UDP-GlcNAc (D).

FIG. 34 shows a high performance liquid chromatogram, which demonstratesa lack of extracellular GnTIII activity (pVA53) in the supernatant. TheN-glycan GlcNAc₂Man₃GlcNAc₂ purified from K3 expressed in YSH-44 strainwas added to: BMMY (A); 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis,Mo.)) in BMMY (B); and the supernatant of YSH-44 transformed with pVA53[YSH-57] (C).

FIG. 35 is a schematic diagram comparing the normal glycosylationpathways in humans and P. pastoris (Panel A) with an engineeredhumanized N-glycosylation pathway in lower eukaryotes (Panel B). Theengineered pathway represents the construction of P. pastoris strainPBP6-5, which after modification with GnTIII becomes P. pastoris strainPBP38.

FIG. 36 is a schematic diagram showing the predominant secretedglycoform produced by each of the designated P. pastoris strains and thegene modification used to engineer each of the strains.

FIG. 37 is a structural representation of the transfer of a GlcNAc tothe oligosaccharide intermediate, GlcNAcMan₅GlcNAc₂, produced onglycoproteins in a lower eukaryotic host cell, as catalyzed by GnTIII.

FIG. 38 is a structural representation of the transfer of a GlcNAc tothe oligosaccharide intermediate, GlcNAcMan₃GlcNAc₂, produced onglycoproteins in a lower eukaryotic host cell, as catalyzed by GnTII,and the subsequent transfer of a GlcNAc to the product of that reaction,GlcNAc₂Man₃GlcNAc₂, as catalyzed by GnTIII.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present invention are generally performed according toconventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

The methods and techniques of the present invention are generallyperformed according to conventional methods well-known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Introduction to Glycobiology,Maureen E. Taylor, Kurt Drickamer, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp. Freehold, N.J.;Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press;Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press;Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).The nomenclatures used in connection with, and the laboratory proceduresand techniques of, molecular and cellular biology, protein biochemistry,enzymology and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

All publications, patents and other references mentioned herein areincorporated by reference.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

As used herein, the term “N-glycan” refers to an N-linkedoligosaccharide, e.g., one that is attached by anasparagine-N-acetylglucosamine linkage to an asparagine residue of apolypeptide. N-glycans have a common pentasaccharide core of Man₃GlcNAc₂(“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers toN-acetyl; GlcNAc refers to N-acetylglucosamine). The term “trimannosecore” used with respect to the N-glycan also refers to the structureMan₃GlcNAc₂ (“Man₃”). The term “pentamannose core” or “Mannose-5 core”or “Man₅” used with respect to the N-glycan refers to the structureMan₅GlcNAc₂. N-glycans differ with respect to the number of branches(antennae) comprising peripheral sugars (e.g., GlcNAc, fucose, andsialic acid) that are attached to the Man₃ core structure. N-glycans areclassified according to their branched constituents (e.g., high mannose,complex or hybrid).

A “high mannose” type N-glycan has five or more mannose residues. A“complex” type N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of the trimannose core. Complex N-glycans may also have galactose(“Gal”) residues that are optionally modified with sialic acid orderivatives (“NeuAc”, where “Neu” refers to neuraminic acid and “Ac”refers to acetyl). A complex N-glycan typically has at least one branchthat terminates in an oligosaccharide such as, for example: NeuNAc-;NeuAcα2-6GalNAcα1-; NeuAcα2-3Galβ1-3GalNAcα1-;NeuAcα2-3/6Galβ1-4GlcNAcβ1-; GlcNAcα1-4Galβ1-(mucins only);Fucα1-2Galβ1-(blood group H). Sulfate esters can occur on galactose,GalNAc, and GlcNAc residues, and phosphate esters can occur on mannoseresidues. NeuAc (Neu: neuraminic acid; Ac: acetyl) can be O-acetylatedor replaced by NeuGl (N-glycolylneuraminic acid). Complex N-glycans mayalso have intrachain substitutions comprising “bisecting” GlcNAc andcore fucose (“Fuc”). A “hybrid” N-glycan has at least one GlcNAc on theterminal of the 1,3 mannose arm of the trimannose core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

The term “predominant” or “predominantly” used with respect to theproduction of N-glycans refers to a structure which represents the majorpeak detected by matrix assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF) analysis.

Abbreviations used herein are of common usage in the art, see, e.g.,abbreviations of sugars, above. Other common abbreviations include“PNGase”, which refers to peptide N-glycosidase F (EC 3.2.2.18); “GlcNAcTr” or “GnT,” which refers to N-acetylglucosaminyl Transferase enzymes;“NANA” refers to N-acetylneuraminic acid.

As used herein, a “humanized glycoprotein” or a “human-likeglycoprotein” refers alternatively to a protein having attached theretoN-glycans having fewer than four mannose residues, and syntheticglycoprotein intermediates (which are also useful and can be manipulatedfurther in vitro or in vivo) having at least five mannose residues.Preferably, glycoproteins produced according to the invention contain atleast 30 mole %, preferably at least 40 mole % and more preferably 50,60, 70, 80, 90, or even 100 mole % of the Man₅GlcNAc₂ intermediate, atleast transiently. This may be achieved, e.g., by engineering a hostcell of the invention to express a “better”, i.e., a more efficientglycosylation enzyme. For example, a mannosidase is selected such thatit will have optimal activity under the conditions present at the sitein the host cell where proteins are glycosylated and is introduced intothe host cell preferably by targeting the enzyme to a host cellorganelle where activity is desired.

The term “enzyme”, when used herein in connection with altering hostcell glycosylation, refers to a molecule having at least one enzymaticactivity, and includes full-length enzymes, catalytically activefragments, chimerics, complexes, and the like. A “catalytically activefragment” of an enzyme refers to a polypeptide having a detectable levelof functional (enzymatic) activity. Enzyme activity is “substantiallyintracellular” when less than 10% of the enzyme activity is measurableoutside the cell compared to that measurable from lysed cells.

A lower eukaryotic host cell, when used herein in connection withglycosylation profiles, refers to any eukaryotic cell which ordinarilyproduces high mannose containing N-glycans, and thus is meant to includesome animal or plant cells and most typical lower eukaryotic cells,including uni- and multicellular fungal and algal cells.

As used herein, the term “secretion pathway” refers to the assembly lineof various glycosylation enzymes to which a lipid-linked oligosaccharideprecursor and an N-glycan substrate are sequentially exposed, followingthe molecular flow of a nascent polypeptide chain from the cytoplasm tothe endoplasmic reticulum (ER) and the compartments of the Golgiapparatus. Enzymes are said to be localized along this pathway. Anenzyme X that acts on a lipid-linked glycan or an N-glycan before enzymeY is said to be or to act “upstream” to enzyme Y; similarly, enzyme Y isor acts “downstream” from enzyme X.

The term “targeting peptide” as used herein refers to nucleotide oramino acid sequences encoding a cellular targeting signal peptide whichmediates the localization. (or retention) of an associated sequence tosub-cellular locations, e.g., organelles.

The term “polynucleotide” or “nucleic acid molecule” refers to apolymeric form of nucleotides of at least 10 bases in length. The termincludes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNAmolecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA orRNA containing non-natural nucleotide analogs, non-nativeinternucleoside bonds, or both. The nucleic acid can be in anytopological conformation. For instance, the nucleic acid can besingle-stranded, double-stranded, triple-stranded, quadruplexed,partially double-stranded, branched, hairpinned, circular, or in apadlocked conformation. The term includes single and double strandedforms of DNA. A nucleic acid molecule of this invention may include bothsense and antisense strands of RNA, cDNA, genomic DNA, and syntheticforms and mixed polymers of the above. They may be modified chemicallyor biochemically or may contain non-natural or derivatized nucleotidebases, as will be readily appreciated by those of skill in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, etc.), chelators, alkylators, and modified linkages (e.g.,alpha anomeric nucleic acids, etc.) Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

Unless otherwise indicated, a “nucleic acid comprising SEQ ID NO:X”refers to a nucleic acid, at least a portion of which has either (i) thesequence of SEQ ID NO:X, or (ii) a sequence complementary to SEQ IDNO:X. The choice between the two is dictated by the context. Forinstance, if the nucleic acid is used as a probe, the choice between thetwo is dictated by the requirement that the probe be complementary tothe desired target.

An “isolated” or “substantially pure” nucleic acid or polynucleotide(e.g., an RNA, DNA or a mixed polymer) is one which is substantiallyseparated from other cellular components that naturally accompany thenative polynucleotide in its natural host cell, e.g., ribosomes,polymerases, and genomic sequences with which it is naturallyassociated. The term embraces a nucleic acid or polynucleotide that (1)has been removed from its naturally occurring environment, (2) is notassociated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (3) is operatively linkedto a polynucleotide which it is not linked to in nature, or (4) does notoccur in nature. The term “isolated” or “substantially pure” also can beused in reference to recombinant or cloned DNA isolates, chemicallysynthesized polynucleotide analogs, or polynucleotide analogs that arebiologically synthesized by heterologous systems.

However, “isolated” does not necessarily require that the nucleic acidor polynucleotide so described has itself been physically removed fromits native environment. For instance, an endogenous nucleic acidsequence in the genome of an organism is deemed “isolated” herein if aheterologous sequence (i.e., a sequence that is not naturally adjacentto this endogenous nucleic acid sequence) is placed adjacent to theendogenous nucleic acid sequence, such that the expression of thisendogenous nucleic acid sequence is altered. By way of example, anon-native promoter sequence can be substituted (e.g., by homologousrecombination) for the native promoter of a gene in the genome of ahuman cell, such that this gene has an altered expression pattern. Thisgene would now become “isolated” because it is separated from at leastsome of the sequences that naturally flank it.

A nucleic acid is also considered “isolated” if it contains anymodifications that do not naturally occur to the corresponding nucleicacid in a genome. For instance, an endogenous coding sequence isconsidered “isolated” if it contains an insertion, deletion or a pointmutation introduced artificially, e.g., by human intervention. An“isolated nucleic acid” also includes a nucleic acid integrated into ahost cell chromosome at a heterologous site, a nucleic acid constructpresent as an episome. Moreover, an “isolated nucleic acid” can besubstantially free of other cellular material, or substantially free ofculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

As used herein, the phrase “degenerate variant” of a reference nucleicacid sequence encompasses nucleic acid sequences that can be translated,according to the standard genetic code, to provide an amino acidsequence identical to that translated from the reference nucleic acidsequence.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over a stretch of at least aboutnine nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides. There are a number of different algorithmsknown in the art which can be used to measure nucleotide sequenceidentity. For instance, polynucleotide sequences can be compared usingFASTA, Gap or Bestfit, which are programs in Wisconsin Package Version10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson (1990) MethodsEnzymol. 183:63-98, incorporated herein by reference in its entirety).For instance, percent sequence identity between nucleic acid sequencescan be determined using FASTA with its default parameters (a word sizeof 6 and the NOPAM factor for the scoring matrix) or using Gap with itsdefault parameters as provided in GCG Version 6.1, herein incorporatedby reference.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 50%, more preferably 60%of the nucleotide bases, usually at least about 70%, more usually atleast about 80%, preferably at least about 90%, and more preferably atleast about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap, as discussed above.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof hybridizes to another nucleic acid, to a strandof another nucleic acid, or to the complementary strand thereof, understringent hybridization conditions. “Stringent hybridization conditions”and “stringent wash conditions” in the context of nucleic acidhybridization experiments depend upon a number of different physicalparameters. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, solvents, the basecomposition of the hybridizing species, length of the complementaryregions, and the number of nucleotide base mismatches between thehybridizing nucleic acids, as will be readily appreciated by thoseskilled in the art. One having ordinary skill in the art knows how tovary these parameters to achieve a particular stringency ofhybridization.

In general, “stringent hybridization” is performed at about 25° C. belowthe thermal melting point (T_(m)) for the specific DNA hybrid under aparticular set of conditions. “Stringent washing” is performed attemperatures about 5° C. lower than the T_(m) for the specific DNAhybrid under a particular set of conditions. The T_(m) is thetemperature at which 50% of the target sequence hybridizes to aperfectly matched probe. See Sambrook et al., supra, page 9.51, herebyincorporated by reference. For purposes herein, “high stringencyconditions” are defined for solution phase hybridization as aqueoushybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours,followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. Itwill be appreciated by the skilled artisan that hybridization at 65° C.will occur at different rates depending on a number of factors includingthe length and percent identity of the sequences which are hybridizing.

The term “mutated” when applied to nucleic acid sequences means thatnucleotides in a nucleic acid sequence may be inserted, deleted orchanged compared to a reference nucleic acid sequence. A singlealteration may be made at a locus (a point mutation) or multiplenucleotides may be inserted, deleted or changed at a single locus. Inaddition, one or more alterations may be made at any number of lociwithin a nucleic acid sequence. A nucleic acid sequence may be mutatedby any method known in the art including but not limited to mutagenesistechniques such as “error-prone PCR” (a process for performing PCR underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. See, e.g., Leung et al. (1989) Technique 1:11-15 andCaldwell and Joyce (1992) PCR Methods Applic. 2:28-33); and“oligonucleotide-directed mutagenesis” (a process which enables thegeneration of site-specific mutations in any cloned DNA segment ofinterest. See, e.g., Reidhaar-Olson et al. (1988) Science 241:53-57).

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Other vectors include cosmids, bacterial artificialchromosomes (BAC) and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments may beligated into the viral genome (discussed in more detail below). Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., vectors having an origin of replication whichfunctions in the host cell). Other vectors can be integrated into thegenome of a host cell upon introduction into the host cell, and arethereby replicated along with the host genome. Moreover, certainpreferred vectors are capable of directing the expression of genes towhich they are operatively linked. Such vectors are referred to hereinas “recombinant expression vectors” (or simply, “expression vectors”).

“Operatively linked” expression control sequences refers to a linkage inwhich the expression control sequence is contiguous with the gene ofinterest to control the gene of interest, as well as expression controlsequences that act in trans or at a distance to control the gene ofinterest.

The term “expression control sequence” as used herein refers topolynucleotide sequences which are necessary to affect the expression ofcoding sequences to which they are operatively linked. Expressioncontrol sequences are sequences which control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is essential forexpression, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a nucleic acid such asa recombinant vector has been introduced. It should be understood thatsuch terms are intended to refer not only to the particular subject cellbut to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell which resides in a living tissueor organism.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that is typically less than about 50 amino acids long and moretypically less than about 30 amino acids long. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” as used herein encompasses bothnaturally-occurring and non-naturally-occurring proteins, and fragments,mutants, derivatives and analogs thereof. A polypeptide may be monomericor polymeric. Further, a polypeptide may comprise a number of differentdomains each of which has one or more distinct activities.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) when it exists in a purity not found in nature,where purity can be adjudged with respect to the presence of othercellular material (e.g., is free of other proteins from the samespecies) (3) is expressed by a cell from a different species, or (4)does not occur in nature (e.g., it is a fragment of a polypeptide foundin nature or it includes amino acid analogs or derivatives not found innature or linkages other than standard peptide bonds). Thus, apolypeptide that is chemically synthesized or synthesized in a cellularsystem different from the cell from which it naturally originates willbe “isolated” from its naturally associated components. A polypeptide orprotein may also be rendered substantially free of naturally associatedcomponents by isolation, using protein purification techniqueswell-known in the art. As thus defined, “isolated” does not necessarilyrequire that the protein, polypeptide, peptide or oligopeptide sodescribed has been physically removed from its native environment.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion compared toa full-length polypeptide. In a preferred embodiment, the polypeptidefragment is a contiguous sequence in which the amino acid sequence ofthe fragment is identical to the corresponding positions in thenaturally-occurring sequence. Fragments typically are at least 5, 6, 7,8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 aminoacids long, more preferably at least 20 amino acids long, morepreferably at least 25, 30, 35, 40 or 45, amino acids, even morepreferably at least 50 or 60 amino acids long, and even more preferablyat least 70 amino acids long.

A “modified derivative” refers to polypeptides or fragments thereof thatare substantially homologous in primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thenative polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those wellskilled in the art. A variety of methods for labeling polypeptides andof substituents or labels useful for such purposes are well-known in theart, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,ligands which bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands whichcan serve as specific binding pair members for a labeled ligand. Thechoice of label depends on the sensitivity required, ease of conjugationwith the primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well-known in the art. See Ausubelet al., Current Protocols in Molecular Biology, Greene PublishingAssociates (1992, and Supplements to 2002), hereby incorporated byreference.

A “polypeptide mutant” or “mutein” refers to a polypeptide whosesequence contains an insertion, duplication, deletion, rearrangement orsubstitution of one or more amino acids compared to the amino acidsequence of a native or wild-type protein. A mutein may have one or moreamino acid point substitutions, in which a single amino acid at aposition has been changed to another amino acid, one or more insertionsand/or deletions, in which one or more amino acids are inserted ordeleted, respectively, in the sequence of the naturally-occurringprotein, and/or truncations of the amino acid sequence at either or boththe amino or carboxy termini. A mutein may have the same but preferablyhas a different biological activity compared to the naturally-occurringprotein.

A mutein has at least 70% overall sequence homology to its wild-typecounterpart. Even more preferred are muteins having 80%, 85% or 90%overall sequence homology to the wild-type protein. In an even morepreferred embodiment, a mutein exhibits 95% sequence identity, even morepreferably 97%, even more preferably 98% and even more preferably 99%overall sequence identity. Sequence homology may be measured by anycommon sequence analysis algorithm, such as Gap or Bestfit.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinity or enzymatic activity, and (5) confer or modify otherphysicochemical or functional properties of such analogs.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, and other unconventional amino acids may also besuitable components for polypeptides of the present invention. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences). In a preferred embodiment, a homologousprotein is one that exhibits 60% sequence homology to the wild typeprotein, more preferred is 70% sequence homology. Even more preferredare homologous proteins that exhibit 80%, 85% or 90% sequence homologyto the wild type protein. In a yet more preferred embodiment, ahomologous protein exhibits 95%, 97%, 98% or 99% sequence identity. Asused herein, homology between two regions of amino acid sequence(especially with respect to predicted structural similarities) isinterpreted as implying similarity in function.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson (1990) Methods Enzymol. 183:63-98).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A preferred algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul et al. (1990) J. Mol. Biol.215:403-410; Gish and States (1993) Nature Genet. 3:266-272; Madden etal. (1996) Meth. Enzymol. 266:131-141; Altschul et al. (1997) NucleicAcids Res. 25:3389-3402; Zhang and Madden (1997) Genome Res. 7:649-656),especially blastp or tblastn (Altschul et al., 1997). Preferredparameters for BLASTp are: Expectation value: 10 (default); Filter: seg(default); Cost to open a gap: 11 (default); Cost to extend a gap: 1(default); Max. alignments: 100 (default); Word size: 11 (default); No.of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (see Pearson(1990) Methods Enzymol. 183:63-98). For example, percent sequenceidentity between amino acid sequences can be determined using FASTA withits default parameters (a word size of 2 and the PAM250 scoring matrix),as provided in GCG Version 6.1, herein incorporated by reference.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements from two or more differentproteins. A fusion protein comprises at least 10 contiguous amino acidsfrom a polypeptide of interest, more preferably at least 20 or 30 aminoacids, even more preferably at least 40, 50 or 60 amino acids, yet morepreferably at least 75, 100 or 125 amino acids. Fusion proteins can beproduced recombinantly by constructing a nucleic acid sequence whichencodes the polypeptide or a fragment thereof in-frame with a nucleicacid sequence encoding a different protein or peptide and thenexpressing the fusion protein. Alternatively, a fusion protein can beproduced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

The term “region” as used herein refers to a physically contiguousportion of the primary structure of a biomolecule. In the case ofproteins, a region is defined by a contiguous portion of the amino acidsequence of that protein.

The term “domain” as used herein refers to a structure of a biomoleculethat contributes to a known or suspected function of the biomolecule.Domains may be co-extensive with regions or portions thereof; domainsmay also include distinct, non-contiguous regions of a biomolecule.Examples of protein domains include, but are not limited to, an Igdomain, an extracellular domain, a transmembrane domain, and acytoplasmic domain.

As used herein, the term “molecule” means any compound, including, butnot limited to, a small molecule, peptide, protein, sugar, nucleotide,nucleic acid, lipid, etc., and such a compound can be natural orsynthetic.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ofthe present invention and will be apparent to those of skill in the art.All publications and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Throughout this specification and claims, the word “comprise” orvariations such as “comprises” or “comprising”, will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Methods for Producing Human-Like Glycoproteins in Lower Eukaryotic HostCells

The invention provides methods for producing a glycoprotein havinghuman-like glycosylation in a non-human eukaryotic host cell. Asdescribed in more detail below, a eukaryotic host cell that does notnaturally express, or which is engineered not to express, one or moreenzymes involved in production of high mannose structures is selected asa starting host cell. Such a selected host cell is engineered to expressone or more enzymes or other factors required to produce human-likeglycoproteins. A desired host strain can be engineered one enzyme ormore than one enzyme at a time. In addition, a nucleic acid moleculeencoding one or more enzymes or activities may be used to engineer ahost strain of the invention. Preferably, a library of nucleic acidmolecules encoding potentially useful enzymes (e.g., chimeric enzymescomprising a catalytically active enzyme fragment ligated in-frame to aheterologous subcellular targeting sequence) is created (e.g., byligation of sub-libraries comprising enzymatic fragments and subcellulartargeting sequences), and a strain having one or more enzymes withoptimal activities or producing the most “human-like” glycoproteins maybe selected by transforming target host cells with one or more membersof the library.

In particular, the methods described herein enable one to obtain, invivo, Man₅GlcNAc₂ structures in high yield, at least transiently, forthe purpose of further modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures inappropriate yields in a host cell, such as a lower eukaryotic organism,generally involves two parallel approaches: (1) reducing high mannosestructures made by endogenous mannosyltransferase activities, if any,and (2) removing 1,2-α-mannose by mannosidases to yield high levels ofsuitable Man₅GlcNAc₂ structures which may be further reacted inside thehost cell to form complex, human-like glycoforms.

Accordingly, a first step involves the selection or creation of aeukaryotic host cell, e.g., a lower eukaryote, capable of producing aspecific precursor structure of Man₅GlcNAc₂ that is able to accept invivo GlcNAc by the action of a GlcNAc transferase I (“GnTI”). In oneembodiment, the method involves making or using a non-human eukaryotichost cell depleted in a 1,6 mannosyltransferase activity with respect tothe N-glycan on a glycoprotein. Preferably, the host cell is depleted inan initiating 1,6 mannosyltransferase activity (see below). Such a hostcell will lack one or more enzymes involved in the production of highmannose structures which are undesirable for producing human-likeglycoproteins.

One or more enzyme activities are then introduced into such a host cellto produce N-glycans within the host cell characterized by having atleast 30 mol % of the Man₅GlcNAc₂ (“Man₅”) carbohydrate structures.Man₅GlcNAc₂ structures are necessary for complex N-glycan formation:Man₅GlcNAc₂ must be formed in vivo in a high yield (e.g., in excess of30%), at least transiently, as subsequent mammalian- and human-likeglycosylation reactions require Man₅GlcNAc₂ or a derivative thereof.

This step also requires the formation of a particular isomeric structureof Man₅GlcNAc₂ within the cell at a high yield. While Man₅GlcNAc₂structures are necessary for complex N-glycan formation, their presenceis by no means sufficient. That is because Man₅GlcNAc₂ may occur indifferent isomeric forms, which may or may not serve as a substrate forGlcNAc transferase I. As most glycosylation reactions are not complete,a particular glycosylated protein generally contains a range ofdifferent carbohydrate structures (i.e., glycoforms) on its surface.Thus, the mere presence of trace amounts (i.e., less than 5%) of aparticular structure like Man₅GlcNAc₂ is of little practical relevancefor producing mammalian- or human-like glycoproteins. It is theformation of a GlcNAc transferase I-accepting Man₅GlcNAc₂ intermediate(FIG. 1B) in high yield (i.e., above 30%), which is required. Theformation of this intermediate is necessary to enable subsequent in vivosynthesis of complex N-glycans on glycosylated proteins of interest(target proteins).

Accordingly, some or all of the Man₅GlcNAc₂ produced by the selectedhost cell must be a productive substrate for enzyme activities along amammalian glycosylation pathway, e.g., can serve as a substrate for aGlcNAc transferase I activity in vivo, thereby forming the human-likeN-glycan intermediate GlcNAcMan₅GlcNAc₂ in the host cell. In a preferredembodiment, at least 10%, more preferably at least 30% and mostpreferably 50% or more of the Man₅GlcNAc₂ intermediate produced in thehost cell of the invention is a productive substrate for GnTI in vivo.It is understood that if, for example, GlcNAcMan₅GlcNAc₂ is produced at10% and Man₅GlcNAc₂ is produced at 25% on a target protein, that thetotal amount of transiently produced Man₅GlcNAc₂ is 35% becauseGlcNAcMan₅GlcNAc₂ is a product of Man₅GlcNAc₂.

One of ordinary skill in the art can select host cells from nature,e.g., existing fungi or other lower eukaryotes that produce significantlevels of Man₅GlcNAc₂ in vivo. As yet, however, no lower eukaryote hasbeen shown to provide such structures in vivo in excess of 1.8% of thetotal N-glycans (see e.g. Maras et al. (1997) Eur. J. Biochem.249:701-707). Alternatively, such host cells may be geneticallyengineered to produce the Man₅GlcNAc₂ structure in vivo. Methods such asthose described in U.S. Pat. No. 5,595,900 may be used to identify theabsence or presence of particular glycosyltransferases, mannosidases andsugar nucleotide transporters in a target host cell or organism ofinterest.

Inactivation of Undesirable Host Cell Glycosylation Enzymes

The methods of the invention are directed to making host cells whichproduce glycoproteins having altered, and preferably human-like,N-glycan structures. In a preferred embodiment, the methods are directedto making host cells in which oligosaccharide precursors are enriched inMan₅GlcNAc₂. Preferably, a eukaryotic host cell is used that does notexpress one or more enzymes involved in the production of high mannosestructures. Such a host cell may be found in nature or may beengineered, e.g., starting with or derived from one of many such mutantsalready described in yeasts. Thus, depending on the selected host cell,one or a number of genes that encode enzymes known to be characteristicof non-human glycosylation reactions will have to be deleted. Such genesand their corresponding proteins have been extensively characterized ina number of lower eukaryotes (e.g., S. cerevisiae, T. reesei, A.nidulans, etc.), thereby providing a list of known glycosyltransferasesin lower eukaryotes, their activities and their respective geneticsequence. These genes are likely to be selected from the group ofmannosyltransferases, e.g. 1,3 mannosyltransferases (e.g. MNN1 in S.cerevisiae) (Graham, 1991), 1,2 mannosyltransferases (e.g. KTR/KREfamily from S. cerevisiae), 1,6 mannosyltransferases (OCH1 from S.cerevisiae), mannosylphosphate transferases and their regulators (MNN4and MNN6 from S. cerevisiae) and additional enzymes that are involved inaberrant, i.e., non-human, glycosylation reactions. Many of these geneshave in fact been deleted individually giving rise to viable phenotypeswith altered glycosylation profiles. Examples are shown in Table 1.

Preferred lower eukaryotic host cells of the invention, as describedherein to exemplify the required manipulation steps, arehypermannosylation-minus (och1) mutants of Pichia pastoris or K. lactis.Like other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂structures in the ER with an α-1,2-mannosidase to yield Man₈GlcNAc₂(FIG. 1A). Through the action of several mannosyltransferases, thisstructure is then converted to hypermannosylated structures(Man_(>9)GlcNAc₂), also known as mannans (FIG. 35A). In addition, it hasbeen found that P. pastoris is able to add non-terminal phosphategroups, through the action of mannosylphosphate transferases, to thecarbohydrate structure. This differs from the reactions performed inmammalian cells, which involve the removal rather than addition ofmannose sugars (FIG. 35A). It is of particular importance to eliminatethe ability of the eukaryotic host cell, e.g., fungus, tohypermannosylate an existing Man₈GlcNAc₂ structure. This can be achievedby either selecting for a host cell that does not hypermannosylate or bygenetically engineering such a cell.

Genes that are involved in the hypermannosylation process have beenidentified, e.g., in Pichia pastoris, and by creating mutations in thesegenes, one can reduce the production of “undesirable” glycoforms. Suchgenes can be identified by homology to existing mannosyltransferases ortheir regulators (e.g., OCH1, MNN4, MNN6, MNN1) found in other lowereukaryotes such as C. albicans, Pichia angusta or S. cerevisiae or bymutagenizing the host strain and selecting for a glycosylation phenotypewith reduced mannosylation. Based on homologies amongst knownmannosyltransferases and mannosylphosphate transferases, one may eitherdesign PCR primers (examples of which are shown in Table 2), or usegenes or gene fragments encoding such enzymes as probes to identifyhomologs in DNA libraries of the target or a related organism.Alternatively, one may identify a functional homolog havingmannosyltransferase activity by its ability to complement particularglycosylation phenotypes in related organisms.

TABLE 2 PCR Primers Target Gene(s) PCR primer A PCR primer B in P.pastoris Homologs ATGGCGAAGGCA TTAGTCCTTCCAA 1,6- OCH1 S. cerevisiae,GATGGCAGT CTTCCTTC mannosyl- Pichia albicans (SEQ ID NO:3) (SEQ ID NO:4)transferase TAYTGGMGNGTN GCRTCNCCCCANC 1,2 KTR/KRE family, GARCYNGAYATHKYTCRTA mannosyl- S. cerevisiae AA (SEQ ID NO:6) transferases (SEQ IDNO:5) Legend: M = A or C, R = A or G, W = A or T, S = C or G, Y = C orT, K = G or T, V = A or C or G, H = A or C or T, D = A or G or T, B = Cor G or T, N = G or A or T or C.

To obtain the gene or genes encoding 1,6-mannosyltransferase activity inP. pastoris, for example, one would carry out the following steps: OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. Mutants of S. cerevisiae areavailable, e.g., from Stanford University, and are commerciallyavailable from ResGen, Invitrogen Corp. (Carlsbad, Calif.). Mutants thatdisplay a normal growth phenotype at elevated temperature, after havingbeen transformed with a P. pastoris DNA library, are likely to carry anOCH1 homolog of P. pastoris. Such a library can be created by partiallydigesting chromosomal DNA of P. pastoris with a suitable restrictionenzyme and, after inactivating the restriction enzyme, ligating thedigested DNA into a suitable vector, which has been digested with acompatible restriction enzyme.

Suitable vectors include, e.g., pRS314, a low copy (CEN6/ARS4) plasmidbased on pBluescript containing the Trp1 marker (Sikorski and Hieter(1989) Genetics 122:19-27) and pFL44S, a high copy (2μ) plasmid based ona modified pUC19 containing the URA3 marker (Bonneaud et al. (1991)Yeast 7:609-615). Such vectors are commonly used by academic researchersand similar vectors are available from a number of different vendors(e.g., Invitrogen (Carlsbad, Calif.); Pharmacia (Piscataway, N.J.); NewEngland Biolabs (Beverly, Mass.)). Further examples include pYES/GS, 2μorigin of replication based yeast expression plasmid from Invitrogen, orYep24 cloning vehicle from New England Biolabs.

After ligation of the chromosomal DNA and the vector, one may transformthe DNA library into a strain of S. cerevisiae with a specific mutationand select for the correction of the corresponding phenotype. Aftersub-cloning and sequencing the DNA fragment that is able to restore thewild-type phenotype, one may use this fragment to eliminate the activityof the gene product encoded by OCH1 in P. pastoris using in vivomutagenesis and/or recombination techniques well-known to those skilledin the art.

Alternatively, if the entire genomic sequence of a particular host cell,e.g., fungus, of interest is known, one may identify such genes simplyby searching publicly available DNA databases, which are available fromseveral sources, such as NCBI, Swissprot. For example, by searching agiven genomic sequence or database with sequences from a known 1,6mannosyltransferase gene (e.g., OCH1 from S. cerevisiae), one canidentify genes of high homology in such a host cell genome which may(but do not necessarily) encode proteins that have1,6-mannosyltransferase activity. Nucleic acid sequence homology aloneis not enough to prove, however, that one has identified and isolated ahomolog encoding an enzyme having the same activity. To date, forexample, no data exist to show that an OCH1 deletion in P. pastoriseliminates the crucial initiating 1,6-mannosyltransferase activity(Martinet et al. (1998) Biotech. Letters 20(12):1171-1177; Contreras etal. WO 02/00856 A2). Thus, no data prove that the P. pastoris OCH1 genehomolog actually encodes that function. That demonstration is providedfor the first time herein.

Homologs to several S. cerevisiae mannosyltransferases have beenidentified in P. pastoris using these approaches. Homologous genes oftenhave similar functions to genes involved in the mannosylation ofproteins in S. cerevisiae and thus their deletion may be used tomanipulate the glycosylation pattern in P. pastoris or, by analogy, inany other host cell, e.g., fungus, plant, insect or animal cells, withsimilar glycosylation pathways.

The creation of gene knock-outs, once a given target gene sequence hasbeen determined, is a well-established technique in the art and can becarried out by one of ordinary skill in the art (see, e.g., Rothstein(1991) Methods in Enzymology 194:281). The choice of a host organism maybe influenced by the availability of good transformation and genedisruption techniques.

If several mannosyltransferases are to be knocked out, the methoddeveloped by Alani and Kleckner (1987) Genetics 116:541-545, forexample, enables the repeated use of a selectable marker, e.g., the URA3marker in yeast, to sequentially eliminate all undesirable endogenousmannosyltransferase activity. This technique has been refined by othersbut basically involves the use of two repeated DNA sequences, flanking acounter selectable marker. For example: URA3 may be used as a marker toensure the selection of a transformants that have integrated aconstruct. By flanking the URA3 marker with direct repeats one may firstselect for transformants that have integrated the construct and havethus disrupted the target gene. After isolation of the transformants,and their characterization, one may counter select in a second round forthose that are resistant to 5-fluoroorotic acid (5-FOA). Colonies thatare able to survive on plates containing 5-FOA have lost the URA3 markeragain through a crossover event involving the repeats mentioned earlier.This approach thus allows for the repeated use of the same marker andfacilitates the disruption of multiple genes without requiringadditional markers. Similar techniques for sequential elimination ofgenes adapted for use in another eukaryotic host cells with otherselectable and counter-selectable markers may also be used.

Eliminating specific mannosyltransferases, such as 1,6mannosyltransferase (OCH1) or mannosylphosphate transferases (MNN6, orgenes complementing lbd mutants) or regulators (MNN4) in P. pastorisenables one to create engineered strains of this organism whichsynthesize primarily Man₈GlcNAc₂ and which can be used to further modifythe glycosylation pattern to resemble more complex glycoform structures,e.g., those produced in mammalian, e.g., human cells. A preferredembodiment of this method utilizes DNA sequences encoding biochemicalglycosylation activities to eliminate similar or identical biochemicalfunctions in P. pastoris to modify the glycosylation structure ofglycoproteins produced in the genetically altered P. pastoris strain.

Methods used to engineer the glycosylation pathway in yeasts asexemplified herein can be used in filamentous fungi to produce apreferred substrate for subsequent modification. Strategies formodifying glycosylation pathways in A. niger and other filamentousfungi, for example, can be developed using protocols analogous to thosedescribed herein for engineering strains to produce human-likeglycoproteins in yeast. Undesired gene activities involved in 1,2mannosyltransferase activity, e.g., KTR/KRE homologs, are modified oreliminated. A filamentous fungus, such as Aspergillus, is a preferredhost because it lacks the 1,6 mannosyltransferase activity and as such,one would not expect a hypermannosylating gene activity, e.g. OCH1, inthis host. By contrast, other desired activities (e.g.,α-1,2-mannosidase, UDP-GlcNAc transporter, glycosyltransferase (GnT),galactosyltransferase (GalT) and sialyltransferase (ST)) involved inglycosylation are introduced into the host using the targeting methodsof the invention.

Engineering or Selecting Hosts Having Diminished Initiating α-1,6Mannosyltransferase Activity

In a preferred embodiment, the method of the invention involves makingor using a host cell which is diminished or depleted in the activity ofan initiating α-1,6-mannosyltransferase, i.e., an initiation specificenzyme that initiates outer chain mannosylation on the α-1,3 arm of theMan₃GlcNAc₂ core structure. In S. cerevisiae, this enzyme is encoded bythe OCH1 gene. Disruption of the OCH1 gene in S. cerevisiae results in aphenotype in which N-linked sugars completely lack the poly-mannoseouter chain. Previous approaches for obtaining mammalian-typeglycosylation in fungal strains have required inactivation of OCH1 (see,e.g., Chiba et al. (1998) J. Biol. Chem. 273:26298-304). Disruption ofthe initiating α-1,6-mannosyltransferase activity in a host cell of theinvention may be optional, however (depending on the selected hostcell), as the Och1p enzyme requires an intact Man₈GlcNAc₂ for efficientmannose outer chain initiation. Thus, host cells selected or producedaccording to this invention which accumulate oligosaccharides havingseven or fewer mannose residues may produce hypoglycosylated N-glycansthat will likely be poor substrates for Och1p (see, e.g., Nakayama etal. (1997) FEBS Lett. 412(3):547-50).

The OCH1 gene was cloned from P. pastoris (Example 1) and K. lactis(Example 9), as described. Using gene-specific primers, a construct wasmade from each clone to delete the OCH1 gene from the genome of P.pastoris and K. lactis (Examples 1 and 9, respectively). Host cellsdepleted in initiating α-1,6-mannosyltransferase activity and engineeredto produce N-glycans having a Man₅GlcNAc₂ carbohydrate structure werethereby obtained (see, e.g., FIGS. 5, 6, and 12; Examples 4 and 9).

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the K. lactis OCH1gene, and homologs, variants and derivatives thereof. The invention alsoprovides nucleic acid molecules that hybridize under stringentconditions to the above-described nucleic acid molecules. Similarly,isolated polypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of theinvention are provided. Also provided are vectors, including expressionvectors, which comprise the above nucleic acid molecules of theinvention, as described further herein. Similarly, host cellstransformed with the nucleic acid molecules or vectors of the inventionare provided.

The invention further provides methods of making or using a non-humaneukaryotic host cell diminished or depleted in an alg gene activity(i.e., alg activities, including equivalent enzymatic activities innon-fungal host cells) and introducing into the host cell at least oneglycosidase activity. In a preferred embodiment, the glycosidaseactivity is introduced by causing expression of one or more mannosidaseactivities within the host cell, for example, by activation of amannosidase activity, or by expression from a nucleic acid molecule of amannosidase activity, in the host cell.

In another embodiment, the method involves making or using a host celldiminished or depleted in the activity of one or more enzymes thattransfer a sugar residue to the 1,6 arm of lipid-linked oligosaccharideprecursors (FIG. 13). A host cell of the invention is selected for or isengineered by introducing a mutation in one or more of the genesencoding an enzyme that transfers a sugar residue (e.g., mannosylates)the 1,6 arm of a lipid-linked oligosaccharide precursor. The sugarresidue is more preferably mannose, is preferably a glucose, GlcNAc,galactose, sialic acid, fucose or GlcNAc phosphate residue. In apreferred embodiment, the activity of one or more enzymes thatmannosylate the 1,6 arm of lipid-linked oligosaccharide precursors isdiminished or depleted. The method may further comprise the step ofintroducing into the host cell at least one glycosidase activity (seebelow).

In yet another embodiment, the invention provides a method for producinga human-like glycoprotein in a non-human host, wherein the glycoproteincomprises an N-glycan having at least two GlcNAcs attached to atrimannose core structure.

In each above embodiment, the method is directed to making a host cellin which the lipid-linked oligosaccharide precursors are enriched inMan_(X)GlcNAc₂ structures, where X is 3, 4 or 5 (FIG. 14). Thesestructures are transferred in the ER of the host cell onto nascentpolypeptide chains by an oligosaccharyl-transferase and may then beprocessed by treatment with glycosidases (e.g., α-mannosidases) andglycosyltransferases (e.g., GnTI) to produce N-glycans havingGlcNAcMan_(X)GlcNAc₂ core structures, wherein X is 3, 4 or 5, and ispreferably 3 (FIGS. 14 and 15). As shown in FIG. 14, N-glycans having aGlcNAcMan_(X)GlcNAc₂ core structure where X is greater than 3 may beconverted to GlcNAcMan₃GlcNAc₂, e.g., by treatment with an α-1,3 and/orα-1,2-1,3 mannosidase activity, where applicable.

Additional processing of GlcNAcMan₃GlcNAc₂ by treatment withglycosyltransferases (e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ corestructures which may then be modified, as desired, e.g., by ex vivotreatment or by heterologous expression in the host cell of a set ofglycosylation enzymes, including glycosyltransferases, sugartransporters and mannosidases (see below), to become human-likeN-glycans. Preferred human-like glycoproteins which may be producedaccording to the invention include those which comprise N-glycans havingseven or fewer, or three or fewer, mannose residues; comprise one ormore sugars selected from the group consisting of galactose, GlcNAc,sialic acid, and fucose; and comprise at least one oligosaccharidebranch comprising the structure NeuNAc-Gal-GlcNAc-Man.

In one embodiment, the host cell has diminished or depletedDol-P-Man:Man₅GlcNAc₂-PP-Dol Mannosyltransferase activity, which is anactivity involved in the first mannosylation step fromMan₅GlcNAc₂-PP-Dol to Man₆GlcNAc₂-PP-Dol at the luminal side of the ER(e.g., ALG3 FIG. 13; FIG. 14). In S. cerevisiae, this enzyme is encodedby the ALG3 gene. As described above, S. cerevisiae cells harboring aleaky alg3-1 mutation accumulate Man₅GlcNAc₂-PP-Dol and cells having adeletion in alg3 appear to transfer Man₅GlcNAc₂ structures onto nascentpolypeptide chains within the ER. Accordingly, in this embodiment, hostcells will accumulate N-glycans enriched in Man₅GlcNAc₂ structures whichcan then be converted to GlcNAc₂Man₃GlcNAc₂ by treatment withglycosidases (e.g., with α-1,2 mannosidase, α-1,3 mannosidase, orα-1,2-1,3 mannosidase activities) and glycosyltransferase activities(e.g., GnTI, GnTII) (FIG. 14; FIG. 35B).

As described in Example 10, degenerate primers were designed based on analignment of Alg3 protein sequences from S. cerevisiae, D. melanogasterand humans (H. sapiens) (FIGS. 16 and 17), and were used to amplify aproduct from P. pastoris genomic DNA. The resulting PCR product was usedas a probe to identify and isolate a P. pastoris genomic clonecomprising an open reading frame (ORF) that encodes a protein having 35%overall sequence identity and 53% sequence similarity to the S.cerevisiae ALG3 gene (FIGS. 18 and 19). This P. pastoris gene isreferred to herein as “PpALG3”. The ALG3 gene was similarly identifiedand isolated from K. lactis (Example 10; FIGS. 20 and 21).

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the P. pastorisALG3 gene (FIG. 18) and the K. lactis ALG3 gene (FIG. 20), and homologs,variants and derivatives thereof. The invention also provides nucleicacid molecules that hybridize under stringent conditions to theabove-described nucleic acid molecules. Similarly, isolated polypeptides(including muteins, allelic variants, fragments, derivatives, andanalogs) encoded by the nucleic acid molecules of the invention areprovided (P. pastoris and K. lactis ALG3 gene products are shown inFIGS. 18 and 20). In addition, also provided are vectors, includingexpression vectors, which comprise a nucleic acid molecule of theinvention, as described further herein.

Using gene-specific primers, a construct was made to delete the PpALG3gene from the genome of P. pastoris (Example 10). This strain was usedto generate a host cell depleted in Dol-P-Man:Man₅GlcNAc₂-PP-DolMannosyltransferase activity and produce lipid-linked Man₅GlcNAc₂-PP-Dolprecursors which are transferred onto nascent polypeptide chains toproduce N-glycans having a Man₅GlcNAc₂ carbohydrate structure.

As described in Example 11, such a host cell may be engineered byexpression of appropriate mannosidases to produce N-glycans having thedesired Man₃GlcNAc₂ core carbohydrate structure. Expression of GnTs inthe host cell (e.g., by targeting a nucleic acid molecule or a libraryof nucleic acid molecules as described below) enables the modified hostcell to produce N-glycans having one or two GlcNAc structures attachedto each arm of the Man3 core structure (i.e., GlcNAc₁Man₃GlcNAc₂,GlcNAc₂Man₃GlcNAc₂, or GlcNAc₃Man₃GlcNAc₂; see FIG. 15). Thesestructures may be processed further using the methods of the inventionto produce human-like N-glycans on proteins which enter the secretionpathway of the host cell.

In a preferred embodiment, the method of the invention involves makingor using a host cell which is both (a) diminished or depleted in theactivity of an alg gene or in one or more activities that mannosylateN-glycans on the α-1,6 arm of the Man₃GlcNAc₂ (“Man3”) core carbohydratestructure; and (b) diminished or depleted in the activity of aninitiating α-1,6-mannosyltransferase, i.e., an initiation specificenzyme that initiates outer chain mannosylation (on the α-1,3 arm of theMan3 core structure). In S. cerevisiae, this enzyme is encoded by theOCH1 gene. Disruption of the och1 gene in S. cerevisiae results in aphenotype in which N-linked sugars completely lack the poly-mannoseouter chain. Previous approaches for obtaining mammalian-typeglycosylation in fungal strains have required inactivation of OCH1 (see,e.g., Chiba et al. (1998) J. Biol. Chem. 273:26298-304). Disruption ofthe initiating α-1,6-mannosyltransferase activity in a host cell of theinvention is optional, however (depending on the selected host cell), asthe Och1p enzyme requires an intact Man₈GlcNAc for efficient mannoseouter chain initiation. Thus, the host cells selected or producedaccording to this invention, which accumulate lipid-linkedoligosaccharides having seven or fewer mannose residues will, aftertransfer, produce hypoglycosylated N-glycans that will likely be poorsubstrates for Och1p (see, e.g., Nakayama et al. (1997) FEBS Lett.412(3):547-50).

Engineering or Selecting Hosts Having N-AcetylglucosaminyltransferaseIII Activity

The invention additionally provides a method for producing a human-likeglycoprotein in a lower eukaryotic host cell by expressing anN-acetylglucosaminyltransferase III activity (including a full-lengthenzyme, homologs, variants, derivatives, and catalytically activefragments thereof). In one embodiment, a host cell (e.g., P. pastoris)is engineered to produce more human-like N-glycans, e.g., by activationof an N-acetylglucosaminyltransferase III activity or by expression froma nucleic acid molecule of an N-acetylglucosaminyltransferase IIIactivity. Using well-known techniques in the art, gene-specific primersare designed to complement the homologous regions of a GnTIII gene,preferably a mammalian GnTIII gene (e.g., mouse GnTIII) (FIG. 24),sequences for which are readily available in the art (e.g., GenbankAccession No. L39373) and are PCR amplified.

In one embodiment, the invention provides a method for producing ahuman-like glycoprotein in a lower eukaryote (e.g., P. pastoris),wherein the glycoprotein comprises an N-glycan exhibiting a bisectingGlcNAc on a trimannose or trimannosyl (Man₃GlcNAc₂) core structure. Inthis embodiment, GlcNAcMan₃GlcNAc₂ (which may be produced by reacting atrimannose core with N-acetylglucosaminyltransferase I (“GnTI”)activity, but which is typically produced by trimming ofGlcNAcMan₅GlcNAc₂ by an α-1,3/α-1,6-mannosidase activity, such asMannosidase II (Hamilton et al. (2003) Science 301:1244-46)) is reactedwith an N-acetylglucosaminyltransferase III activity to produce abisected GlcNAc₂Man₃GlcNAc₂. Accordingly, the invention provides GnTIIIactivity, which transfers β-1,4 GlcNAc onto substrates that are capableof accepting the bisecting GlcNAc in lower eukaryotes.

In another embodiment, the invention provides a method for producing ahuman-like glycoprotein in a lower eukaryote (e.g., P. pastoris),wherein the glycoprotein comprises an N-glycan exhibiting a bisectingGlcNAc on a trimannose or trimannosyl (Man₃GlcNAc₂) core structurehaving at least two GlcNAcs attached to the trimannose core. In thisembodiment, Man₃GlcNAc₂ is reacted with a GnTI activity and then with anN-acetylglucosaminyltransferase II (“GnTII”) activity and a GnTIIIactivity (in either order) to produce a bisected GlcNAc₃Man₃GlcNAc₂(FIG. 38). It should be appreciated that the bisected trimannosyl corestructure of this embodiment may also contain an additional mannosylgroup in place of a GlcNAc residue. For example, GlcNAcMan₄GlcNAc₂ maybe reacted with a GnTIII activity to produce a bisectedGlcNAc₂Man₄GlcNAc₂.

The invention also provides a method for producing a more human-likeglycoprotein in a lower eukaryote (e.g. P. pastoris), wherein theglycoprotein produced comprises an N-glycan having at least two GlcNAcsattached to a pentamannose core structure (Man₅GlcNAc₂) and whichexhibits a bisected N-glycan. Accordingly, in this embodiment, apentamannose core structure (Man₅GlcNAc₂) is reacted with GnTIIIactivity to produce a bisected GlcNAcMan₅GlcNAc₂ and GlcNAc₂Man₅GlcNAc₂structure.

In an alternative embodiment, a pentamannose core structure produced viathe mutation of och1 and alg3 genes is reacted with α1,2-mannosidase,GnTI, GnTII and GnTIII activities and UDP-GlcNAc to produce a bisectedGlcNAc₃Man₃GlcNAc₂ glycan (FIG. 35B). In another embodiment, apentamannose core structure is reacted with GnTI and GnTIII activities(in either order or in combination) to produce a bisectedGlcNAc₂Man₅GlcNAc₂ structure (FIG. 37).

In a more preferred embodiment, using the combinatorial DNA librarymethod of the invention, as described below, a pVA53 constructcomprising the S. cerevisiae MNN2(s) leader (GenBank Accession No.NP_(—)009571) fused to a catalytically active GnTIII domain from mouse(GnTIII Δ32) is expressed in a P. pastoris strain YSH-1 (Example 13)thereby producing N-glycans having a bisected GlcNAc₂Man₅GlcNAc₂structure (Example 20). FIG. 26 (bottom) displays the MALDI-TOF spectrumof N-glycans released from a kringle 3 protein expressed in theabove-mentioned strain, which is designated PBP26 (FIG. 36), exhibitinga predominant peak at 1666 m/z [a], which corresponds to bisectedGlcNAc₂Man₅GlcNAc₂. (For comparison, FIG. 26 (top) displays theMALDI-TOF spectrum of N-glycans released from a kringle 3 proteinexpressed in strain YSH-1 lacking the pVA53 construct. The predominantpeak at 1461 m/z [d] corresponds to the unmodified glycan:GlcNAcMan₅GlcNAc₂.) Accordingly, in one embodiment, a host of thepresent invention is characterized by its ability to produce, at leasttransiently, N-glycans which exhibit at least 50 mole % of aGlcNAc₂Man₅GlcNAc₂ or at least 50 mole % of a GlcNAc₂Man₃GlcNAc₂structure having a bisecting GlcNAc. The mole percent of the glycans isin reference to percent of total neutral glycans as detected byMALDI-TOF. It is understood that if, for example, GlcNAc₂Man₃GlcNAc₂having a bisecting GlcNAc is produced at 20% and GlcNAc₃Man₃GlcNAc₂ isproduced at 25% on a target protein, the total amount of transientlyproduced GlcNAc₂Man₃GlcNAc₂ having a bisecting GlcNAc is 45%, becauseGlcNAc₃Man₃GlcNAc₂ is a product of a GlcNAc₂Man₃GlcNAc₂ having abisecting GlcNAc further reacted with GnTII.

Similarly, in another embodiment, a pVA55 construct comprising the S.cerevisiae MNN2(1) leader (GenBank Accession No. NP_(—)009571) fused toa catalytically active GnTIII domain from mouse (GnTIII Δ32) isexpressed in a P. pastoris strain (YSH-1) thereby producing N-glycansGlcNAcMan₅GlcNAc₂ and bisected N-glycans GlcNAc₂Man₅GlcNAc₂ structure.As shown in FIG. 27 (bottom), these structures correspond to peaks at1463 m/z and 1667 m/z, respectively. (For comparison, FIG. 27 (top)displays the MALDI-TOF spectrum of N-glycans released from a kringle 3protein expressed in strain YSH-1 lacking the pVA53 construct. Thepredominant peak corresponds to unmodified GlcNAcMan₅GlcNAc₂ at 1461 m/z[d].) Accordingly, in another embodiment, a host of the presentinvention is characterized by its ability to produce, at leasttransiently, N-glycans which exhibit at least 20 mole % of aGlcNAc₂Man₅GlcNAc₂ or at least 20 mole % of a GlcNAc₂Man₃GlcNAc₂structure having a bisecting GlcNAc.

In an even more preferred embodiment, a pVA53 construct comprising theS. cerevisiae MNN2(s) leader (GenBank Accession No. NP_(—)009571) fusedto a catalytically active GnTIII domain from mouse (GnTIII Δ32) isexpressed in a P. pastoris strain YSH-44 (Example 15) thereby producingN-glycans having a bisected GlcNAc₃Man₃GlcNAc₂ structure (Example 20).FIG. 30 displays the MALDI-TOF spectrum of N-glycans released from akringle 3 protein expressed in the above-mentioned strain designated asYSH-57, exhibiting a predominant peak at 1542 m/z [y], which correspondsto the bisected glycan GlcNAc₃Man₃GlcNAc₂. (For comparison, FIG. 29displays the MALDI-TOF spectrum of N-glycans released from a kringle 3protein expressed in strain YSH-44 lacking the pVA53 construct. Thepredominant peak at 1356 m/z [x] in FIG. 29 corresponds to theunmodified glycan: GlcNAc₂Man₃GlcNAc₂.) Accordingly, in one embodiment,a host of the present invention is characterized by its ability toproduce, at least transiently, N-glycans which exhibit at least 80 mole% of a GlcNAc₃Man₃GlcNAc₂ structure having a bisecting GlcNAc. The molepercent of the glycans is in reference to percent of total neutralglycans as detected by MALDI-TOF.

Alternatively, in another embodiment, a pVA53 construct comprising theS. cerevisiae MNN2(s) leader (GenBank Accession No. NP_(—)009571) fusedto a catalytically active GnTIII domain from mouse (GnTIII Δ32) isexpressed in a P. pastoris strain (PBP6-5) (Example 11) therebyproducing N-glycans having a GlcNAc₂Man₃GlcNAc₂ and a bisectedGlcNAc₃Man₃GlcNAc₂ structure. As shown in FIG. 32, these structurescorrespond to peaks at 1340 m/z and 1543 m/z, respectively. Accordingly,in another embodiment, a host of the present invention is characterizedby its ability to produce, at least transiently, N-glycans which exhibitat least 20 mole % of a GlcNAc₃Man₃GlcNAc₂ structure having a bisectingGlcNAc in an alg3 mutant host cell.

The invention provides methods for producing a human-like glycoproteinin a lower eukaryote, wherein the glycoprotein comprises a Man₅GlcNAc₂core structure or a Man₃GlcNAc₂ core structure, and wherein the corestructure is further modified by two or more GlcNAcs. In someembodiments of the invention, 10% or more of the core structures aremodified by the two or more GlcNAcs. In other preferred embodiments,20%, 30%, 40%, 50%, 60%, 70%, 80% or even more of the core structuresare so modified. In a highly preferred embodiment, one of the GlcNAcs isa bisecting GlcNAc.

In another aspect of the invention, a combinatorial nucleic acid librarywhich encodes at least one GnTIII catalytic domain is used to express aGnTIII activity in a lower eukaryotic host cell (Example 18).Preferably, a library of the invention comprises a sublibrary of leadersequences fused in frame to a single nucleic acid molecule or asublibrary of nucleic acid molecules comprising GnTIII sequences, one ormore of which encode a catalytic domain having GnTIII activity in thehost cell. Alternatively, a single nucleic acid molecule or a sublibraryof nucleic acid molecules comprising leader sequences is fused in frameto a sublibrary of nucleic acid molecules comprising GnTIII sequences,one or more of which encode a catalytic domain having GnTIII activity inthe host cell. (See below.) Expression of these and other suchcombinatorial libraries is performed in a host cell which expresses atarget glycoprotein whose N-glycan structures are analyzed to determinewhether and how much GnTIII is expressed. A wide range of catalyticallyactive GnTIII enzymes may be produced in a host cell using the methodsand libraries of the invention. It is this aspect of the invention thatallows a skilled artisan to create and delinate between GnTIII enzymeshaving little or no activity and those enzymes that are activelyexpressed and which produce predominant levels of a desired bisectedoligosaccharide intermediate such as GlcNAc₂Man₅GlcNAc₂,GlcNAc₃Man₃GlcNAc₂ or GlcNAc₂Man₃GlcNAc₂ in the host cells.

As described further below, the proper targeting of an enzymeresponsible for a given step in the glycosylation pathway to theappropriate subcellular location and the sufficiency of the enzyme'sactivity at the particular pH of that subcellular location are importantfactors in the production of glycoproteins having N-glycans with thedesired structures. The use of combinatorial libraries of fusionproteins to generate diverse populations of enzyme chimeras and thescreening of these libraries in transformed cells provides a powerfulmethod to identify host strains with the activity of interest in theappropriate location. In preferred embodiments of the invention, theenzyme activity is located such that an N-glycan-containing glycoproteinexpressed in the cell is capable of reacting with the activity duringthe secretion process.

Not all combinations of leader/catalytic domains produce desired enzymeactivities however. A wide variety of leader/catalytic domaincombinations is created, only a few of which may be useful in producingthe presently desired intermediates. The present invention,nevertheless, encompasses even those combinations that do not presentlyexhibit a desired enzymatic activity in the exemplified host cell. FIG.28 (bottom) shows a pVB51 construct comprising the K. lactis GNT(s)leader (GenBank Accession No. AF106080) fused to a catalytically activeGnTIII domain from mouse (GnTIII A32) expressed in a P. pastoris strainYSH-1, which does not readily exhibit GnTIII activity. (For comparison,FIG. 28 (top) displays the MALDI-TOF spectrum of N-glycans released froma kringle 3 protein expressed in strain YSH-1 lacking the pVA53construct. The predominant peak corresponds to unmodifiedGlcNAcMan₅GlcNAc₂ at 1461 m/z.) The predominant peak in FIG. 28 (bottom)at 1463 m/z, which correlates to the mass of GlcNAcMan₅GlcNAc₂, isobserved. A second peak at 1726 m/z, which does not correlate to themass of GlcNAc₂Man₅GlcNAc₂ is also observed. It is contemplated thatthese and other such combinations may be useful, with or without slightmodifications using techniques well known in the art, when they areexpressed, e.g., in other host cells including those which have beenmodified to produce human-like glycoforms.

The use of combinatorial libraries to generate diverse populations ofenzyme chimeras and the screening of these libraries in transformedcells further allows strains to be identified in which the enzymeactivity is substantially intracellular. Example 6, below, provides anexample of assay conditions useful for measuring extracellularα-1,2-mannosidase activity. Examples 22 and 23 also provide examples ofassays for glycosyltransferase activity (GnTIII) in the medium. See alsoTable 9, below, and Choi et al. (20033 Proc. Natl. Acad. Sci. U.S.A.100(9):5022-27. For purposes of the invention, an enzyme activity issubstantially intracellular when less than 10% of the enzyme activity ismeasurable in the extracellular medium.

As described in Examples 11, 12, 13, 14, 15, and 19-21, a host cell maybe engineered by the expression of appropriate glycosyltransferases(e.g., N-acetylglucosaminyltransferase) to produce N-glycans having thedesired carbohydrate structures (e.g., GlcNAc₂Man₃GlcNAc₂,GlcNAc₃Man₃GlcNAc₂). Expression of GnTs in the host cell (e.g., bytargeting a nucleic acid molecule or a library of nucleic acid moleculesas described below and in Choi et al. (2003) Proc. Natl. Acad. Sci.US.A. 100(9):5022-27 and WO 02/00879) enables the modified host cell toproduce N-glycans having the bisecting GlcNAc on the middle mannose.These structures may be processed further using the methods of theinvention to produce human-like N-glycans on proteins which enter thesecretion pathway of the host cell.

In a more preferred embodiment, co-expression of appropriateUDP-sugar-transporter(s) and transferase(s) will cap the terminal α-1,6and α-1,3 residues as well as the middle mannose with GlcNAc, resultingin the precursor for mammalian-type complex (e.g. GlcNAc₃Man₃GlcNAc₂)and hybrid N-glycosylation. These peptide-bound N-linked oligosaccharidechains then serve as a precursor for further modification to amammalian-type oligosaccharide structure. Subsequent expression ofgalactosyl-tranferases and genetically engineering the capacity totransfer sialylic acid to the termini (see FIG. 1B) will produce amammalian-type (e.g., human-like) N-glycan structure.

Host Cells of the Invention

A preferred host cell of the invention is a lower eukaryotic cell, e.g.,yeast, a unicellular and multicellular or filamentous fungus. However, awide variety of host cells are envisioned as being useful in the methodsof the invention. Plant cells or insect cells, for instance, may beengineered to express a human-like glycoprotein according to theinvention. Likewise, a variety of non-human, mammalian host cells may bealtered to express more human-like or otherwise altered glycoproteinsusing the methods of the invention. As one of skill in the art willappreciate, any eukaryotic host cell (including a human cell) may beused in conjunction with a library of the invention to express one ormore chimeric proteins which is targeted to a subcellular location,e.g., organelle, in the host cell where the activity of the protein ismodified, and preferably is enhanced. Such a protein is preferably—butneed not necessarily be—an enzyme involved in protein glycosylation, asexemplified herein. It is envisioned that any protein coding sequencemay be targeted and selected for modified activity in a eukaryotic hostcell using the methods described herein.

Lower eukaryotes that are able to produce glycoproteins having theattached N-glycan Man₅GlcNAc₂ are particularly useful because (a)lacking a high degree of mannosylation (e.g., greater than 8 mannosesper N-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to form an even more human-likeglycoform, e.g., by the action of GlcNAc transferase I (FIG. 1B; β1,2GnTI) to form GlcNAcMan₅GlcNAc₂. A yield is obtained of greater than 30mole %, more preferably a yield of 50, 60, 70, 80, 90, or even 100 mole%, glycoproteins with N-glycans having a Man₅GlcNAc₂ structure. In apreferred embodiment, more than 50% of the Man₅GlcNAc₂ structure isshown to be a substrate for a GnTI activity and can serve as such asubstrate in vivo.

Preferred lower eukaryotes of the invention include but are not limitedto: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichiakoclamae, Pichia membranaefaciens, Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reseei, Chrysosporiumlucknowense, Fusarium sp. Fusarium gramineum, Fusarium venenatum, andNeurospora crassa.

In each above embodiment, the method is directed to making a host cellin which the oligosaccharide precursors are enriched in Man₅GlcNAc₂.These structures are desirable because they may then be processed bytreatment in vitro, for example, using the method of Maras andContreras, U.S. Pat. No. 5,834,251. In a preferred embodiment, however,precursors enriched in Man₅GlcNAc₂ are processed by at least one furtherglycosylation reaction in vivo—with glycosidases (e.g., α-mannosidases)and glycosyltransferases (e.g., GnTI)—to produce human-like N-glycans.Oligosaccharide precursors enriched in Man₅GlcNAc₂, for example, arepreferably processed to those having GlcNAcMan_(X)GlcNAc₂ corestructures, wherein X is 3, 4 or 5, and is preferably 3. N-glycanshaving a GlcNAcMan_(X)GlcNAc₂ core structure where X is greater than 3may be converted to GlcNAcMan₃GlcNAc₂, e.g., by treatment with an α-1,3and/or α-1,6 mannosidase activity, where applicable. Additionalprocessing of GlcNAcMan₃GlcNAc₂ by treatment with glycosyltransferases(e.g., GnTII) produces GlcNAc₂Man₃GlcNAc₂ core structures which may thenbe modified, as desired, e.g., by ex vivo treatment or by heterologousexpression in the host cell of additional glycosylation enzymes,including glycosyltransferases, sugar transporters and mannosidases (seebelow), to become human-like N-glycans.

Preferred human-like glycoproteins which may be produced according tothe invention include those which comprise N-glycans having seven orfewer, or three or fewer, mannose residues; and which comprise one ormore sugars selected from the group consisting of galactose, GlcNAc,sialic acid, and fucose.

Another preferred non-human host cell of the invention is a lowereukaryotic cell, e.g., a unicellular or filamentous fungus, which isdiminished or depleted in the activity of one or more alg geneactivities (including an enzymatic activity which is a homolog orequivalent to an alg activity). Another preferred host cell of theinvention is diminished or depleted in the activity of one or moreenzymes (other than alg activities) that mannosylate the α-1,6 arm of alipid-linked oligosaccharide structure.

While lower eukaryotic host cells are preferred, a wide variety of hostcells having the aforementioned properties are envisioned as beinguseful in the methods of the invention. Plant cells, for instance, maybe engineered to express a human-like glycoprotein according to theinvention. Likewise, a variety of non-human, mammalian host cells may bealtered to express more human-like glycoproteins using the methods ofthe invention. An appropriate host cell can be engineered, or one of themany such mutants already described in yeasts may be used. A preferredhost cell of the invention, as exemplified herein, is ahypermannosylation-minus (OCH1) mutant in Pichia pastoris which hasfurther been modified to delete the alg3 gene.

The invention additionally provides lower eukaryotic host cells capableof producing glycoproteins having bisected N-glycans, such as bisectedGlcNAcMan₅GlcNAc₂, GlcNAc₂Man₅GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, and,preferably, GlcNAc₃Man₃GlcNAc₂. In a preferred embodiment of theinvention, the host cells comprise a GnTIII activity. In a morepreferred embodiment, the host cells further comprise one or moreactivities selected from: GnTI, GnTII, GnTIV, and GnTV. Preferred hostcells express GnTI, GnTII, and GnTIII. Other preferred host cellsadditionally express GnTIV and/or GnTV. Even more preferably, the one ormore GnT activities of the host cells are substantially intracellular.

Thus, in preferred embodiments of the invention, host cells comprisingthe one or more GnT activities produce N-glycans comprising structures,including but not limited to GlcNAcMan₃GlcNAc₂, GlcNAcMan₄GlcNAc₂, orGlcNAcMan₅GlcNAc₂, that are capable of reacting with a GnTIII enzymeactivity to produce corresponding bisected N-glycans. The enzymeactivities thereby convert glycoproteins containing these N-glycans intoforms with new and more desirable properties. Because GnTIII iscurrently understood to inhibit additional GnT activity in mammaliancells, the skilled artisan should appreciate that sequentialglycosylation reaction may or may not be of importance. The presentinvention contemplates, however, the addition of GnTI and GnTIII ineither order or together. It should also be understood that other enzymeactivities within the cell, such as, e.g., one or more desiredmannosidase activities (e.g., α 1,2 mannosidase, Mannosidase I,Mannosidase II), may act in concert with the GnT activities to generateyet other human-like glycoproteins of interest (see FIG. 1B).

In a preferred embodiment, a mannosidase II or a catalytically activefragment thereof is introduced into the host cell to trim the α1,3 andα1,6 mannose containing arms of a bisected pentamannose core structuresuch as GlcNAc₂Man₅GlcNAc₂. The resulting glycans (e.g., bisectedGlcNAc₂Man₄GlcNAc₂ and GlcNAc₂Man₃GlcNAc₂) are preferred substrates forsubsequent human-like N-glycan modification.

In another embodiment of the invention, the host cells comprise aMan₅GlcNAc₂ core structure or a Man₃GlcNAc₂ core structure modified bytwo or more GlcNAcs. It should be understood that either core structuremay include further modifications in addition to the modification byGlcNAc. Preferably, 10% or more of the core structures are modified byGlcNAcs. Most preferably, 20%, 30%, 40%, 50%, 60%, 70%, 80% or even moreof the core structures contain the GlcNAc modification.

Formation of Complex N-Glycans

Formation of complex N-glycan synthesis is a sequential process by whichspecific sugar residues are removed and attached to the coreoligosaccharide structure. In higher eukaryotes, this is achieved byhaving the substrate sequentially exposed to various processing enzymes.These enzymes carry out specific reactions depending on their particularlocation within the entire processing cascade. This “assembly line”consists of ER, early, medial and late Golgi, and the trans Golginetwork all with their specific processing environment. To re-create theprocessing of human glycoproteins in the Golgi and ER of lowereukaryotes, numerous enzymes (e.g., glycosyltransferases, glycosidases,phosphatases and transporters) have to be expressed and specificallytargeted to these organelles, and preferably, in a location so that theyfunction most efficiently in relation to their environment as well as toother enzymes in the pathway.

Because one goal of the methods described herein is to achieve a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the hostcell chromosome involves careful planning. As described above, one ormore genes which encode enzymes known to be characteristic of non-humanglycosylation reactions are preferably deleted. The engineered cellstrain is transformed with a range of different genes encoding desiredactivities, and these genes are transformed in a stable fashion, therebyensuring that the desired activity is maintained throughout thefermentation process.

Any combination of the following enzyme activities may be engineeredsingly or multiply into the host using methods of the invention:sialyltransferases, mannosidases, fucosyltransferases,galactosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn- and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose andCMP-N-acetylneuraminic acid. Preferably, enzyme activities areintroduced on one or more nucleic acid molecules (see also below).Nucleic acid molecules may be introduced singly or multiply, e.g., inthe context of a nucleic acid library such as a combinatorial library ofthe invention. It is to be understood, however, that single or multipleenzymatic activities may be introduced into a host cell in any fashion,including but not limited to protein delivery methods and/or by use ofone or more nucleic acid molecules without necessarily using a nucleicacid library or combinatorial library of the invention.

Expression of Glycosyltransferases to Produce Complex N-Glycans:

With DNA sequence information, the skilled artisan can clone DNAmolecules encoding GnT activities (e.g., Example 3, 8, 11, 15, and 18).Using standard techniques well-known to those of skill in the art,nucleic acid molecules encoding GnTI, II, III, IV or V (or encodingcatalytically active fragments thereof) may be inserted into appropriateexpression vectors under the transcriptional control of promoters andother expression control sequences capable of driving transcription in aselected host cell of the invention, e.g., a fungal host such as Pichiasp., Kluyveromyces sp. and Aspergillus sp., as described herein, suchthat one or more of these mammalian GnT enzymes may be activelyexpressed in a host cell of choice for production of a human-likecomplex glycoprotein (e.g., Examples 8, 20, and 21).

Several individual glycosyltransferases have been cloned and expressedin S. cerevisiae (GalT, GnTI), Aspergillus nidulans (GnTI) and otherfungi, without however demonstrating the desired outcome of“humanization” on the glycosylation pattern of the organisms (Yoshida etal. (1999) Glycobiology 9(1):53-8; Kalsner et al. (1995) Glycoconj. J12(3):360-370). It was speculated that the carbohydrate structurerequired to accept sugars by the action of such glycosyltransferases wasnot present in sufficient amounts, which most likely contributed to thelack of complex N-glycan formation.

A preferred method of the invention provides the functional expressionof a GnT, such as GnTI, GnTII, and GnTIII, in the early, medial or lateGolgi apparatus, as well as ensuring a sufficient supply of UDP-GlcNAc(e.g., by expression of a UDP-GlcNAc transporter; see Examples below).

Methods for Providing Sugar Nucleotide Precursors to the GolgiApparatus:

For a glycosyltransferase to function satisfactorily in the Golgi, theenzyme requires a sufficient concentration of an appropriate nucleotidesugar, which is the high-energy donor of the sugar moiety added to anascent glycoprotein. In humans, the full range of nucleotide sugarprecursors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, etc.) are generallysynthesized in the cytosol and transported into the Golgi, where theyare attached to the core oligosaccharide by glycosyltransferases.

To replicate this process in non-human host cells, such as lowereukaryotes, sugar nucleoside specific transporters have to be expressedin the Golgi to ensure adequate levels of nucleoside sugar precursors(Sommers and Hirschberg (1981) J. Cell Biol. 91(2):A406-A406; Sommersand Hirschberg (1982) J. Biol. Chem. 257(18):811-817; Perez andHirschberg (1987) Methods in Enzymology 138:709-715). Nucleotide sugarsmay be provided to the appropriate compartments, e.g., by expressing inthe host microorganism an exogenous gene encoding a sugar nucleotidetransporter. The choice of transporter enzyme is influenced by thenature of the exogenous glycosyltransferase being used. For example, aGlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, and asialyltransferase may require a CMP-sialic acid transporter.

The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g., to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g., UDP,GDP, or CMP. Nucleoside monophosphates can be directly exported from theGolgi in exchange for nucleoside triphosphate sugars by an antiportmechanism. Accumulation of a nucleoside diphosphate, however, inhibitsthe further activity of a glycosyltransferase. As this reaction appearsto be important for efficient glycosylation, it is frequently desirableto provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate.

Suitable transporter enzymes, which are typically of mammalian origin,are described below. Such enzymes may be engineered into a selected hostcell using the methods of the invention.

In another example, α2,3- or α2,6-sialyltransferase caps galactoseresidues with sialic acid in the trans-Golgi and TGN of humans leadingto a mature form of the glycoprotein (FIG. 1B). To reengineer thisprocessing step into a metabolically engineered yeast or fungus willrequire (1) α2,3- or α2,6-sialyltransferase activity and (2) asufficient supply of CMP-N-acetyl neuraminic acid, in the late Golgi ofyeast. To obtain sufficient a 2,3-sialyltransferase activity in the lateGolgi, for example, the catalytic domain of a known sialyltransferase(e.g. from humans) has to be directed to the late Golgi in fungi (seeabove). Likewise, transporters have to be engineered to allow thetransport of CMP-N-acetyl neuraminic acid into the late Golgi. There iscurrently no indication that fungi synthesize or can even transportsufficient amounts of CMP-N-acetyl neuraminic acid into the Golgi.Consequently, to ensure the adequate supply of substrate for thecorresponding glycosyltransferases, one has to metabolically engineerthe production of CMP-sialic acid into the fungus.

UDP-N-Acetylglucosamine

The cDNA of human UDP-N-acetylglucosamine transporter, which wasrecognized through a homology search in the expressed sequence tagsdatabase (dbEST), has been cloned (Ishida (1999) J. Biochem.126(1):68-77). The mammalian Golgi membrane transporter forUDP-N-acetylglucosamine was cloned by phenotypic correction with cDNAfrom canine kidney cells (MDCK) of a recently characterizedKluyveromyces lactis mutant deficient in Golgi transport of the abovenucleotide sugar (Guillen et al. (1998) Proc. Natl. Acad. Sci. USA95(14):7888-7892). Results demonstrate that the mammalian GolgiUDP-GlcNAc transporter gene has all of the necessary information for theprotein to be expressed and targeted functionally to the Golgi apparatusof yeast and that two proteins with very different amino acid sequencesmay transport the same solute within the same Golgi membrane (Guillen etal. (1998) Proc. Natl. Acad. Sci. USA 95(14):7888-7892).

Accordingly, one may incorporate the expression of a UDP-GlcNActransporter in a host cell by means of a nucleic acid construct whichmay contain, for example: (1) a region by which the transformedconstruct is maintained in the cell (e.g., origin of replication or aregion that mediates chromosomal integration), (2) a marker gene thatallows for the selection of cells that have been transformed, includingcounterselectable and recyclable markers such as ura3 or T-urf13(Soderholm et al. (2001) Biotechniques 31(2):306-10) or other wellcharacterized selection-markers (e.g., his4, bla, Sh ble etc.), (3) agene or fragment thereof encoding a functional UDP-GlcNAc transporter(e.g., from K. lactis, (Abeijon, (1996) Proc. Natl. Acad. Sci. USA.93:5963-5968), or from H. sapiens (Ishida et al. (1996) J. Biochem.(Tokyo) 120(6):1074-8), and (4) a promoter activating the expression ofthe above mentioned localization/catalytic domain fusion constructlibrary.

GDP-Fucose

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli and Hirschberg (1999) J. Biol. Chem.274(50):35596-35600. The corresponding gene has not been identified,however, N-terminal sequencing can be used for the design ofoligonucleotide probes specific for the corresponding gene. Theseoligonucleotides can be used as probes to clone the gene encoding forGDP-fucose transporter.

UDP-Galactose

Two heterologous genes, gma12(+) encoding alpha1,2-galactosyltransferase (alpha 1,2 GalT) from Schizosaccharomycespombe and (hUGT2) encoding human UDP-galactose (UDP-Gal) transporter,have been functionally expressed in S. cerevisiae to examine theintracellular conditions required for galactosylation. Correlationbetween protein galactosylation and UDP-galactose transport activityindicated that an exogenous supply of UDP-Gal transporter, rather thanalpha 1,2 GalT played a key role for efficient galactosylation in S.cerevisiae (Kainuma (1999) Glycobiology 9(2):133-141). Likewise, anUDP-galactose transporter from S. pombe was cloned (Segawa (1999) FEBSLetters 451 (3):295-298).

CMP-N-Acetylneuraminic Acid (CMP-Sialic Acid)

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells (Aoki et al. (1999) J. Biochem. (Tokyo)126(5):940-50; Eckhardt et al. (1997) Eur. J Biochem. 248(1):187-92).The functional expression of the murine CMP-sialic acid transporter wasachieved in Saccharomyces cerevisiae (Berninsone et al. (1997) J. Biol.Chem. 272(19):12616-9). Sialic acid has been found in some fungi,however it is not clear whether the chosen host system will be able tosupply sufficient levels of CMP-Sialic acid. Sialic acid can be eithersupplied in the medium or alternatively fungal pathways involved insialic acid synthesis can also be integrated into the host genome.

Expression of Diphosphatases:

When sugars are transferred onto a glycoprotein, either a nucleosidediphosphate or monophosphate is released from the sugar nucleotideprecursors. While monophosphates can be directly exported in exchangefor nucleoside triphosphate sugars by an antiport mechanism,diphosphonucleosides (e.g. GDP) have to be cleaved by phosphatases (e.g.GDPase) to yield nucleoside monophosphates and inorganic phosphate priorto being exported. This reaction appears to be important for efficientglycosylation, as GDPase from S. cerevisiae has been found to benecessary for mannosylation. However, the enzyme only has 10% of theactivity towards UDP (Berninsone et al. (1994) J. Biol. Chem.269(1):207-211). Lower eukaryotes often do not have UDP-specificdiphosphatase activity in the Golgi as they do not utilize UDP-sugarprecursors for glycoprotein synthesis in the Golgi. Schizosaccharomycespombe, a yeast which adds galactose residues to cell wallpolysaccharides (from UDP-galactose), was found to have specific UDPaseactivity, further suggesting the requirement for such an enzyme(Berninsone et al. (1994) J. Biol. Chem. 269(1):207-211). UDP is knownto be a potent inhibitor of glycosyltransferases and the removal of thisglycosylation side product is important to prevent glycosyltransferaseinhibition in the lumen of the Golgi (Khatara et al. (1974) Eur. J.Biochem. 44:537-560).

Methods for Altering N-Glycans in a Host by Expressing a TargetedEnzymatic Activity from a Nucleic Acid Molecule

The present invention further provides a method for producing ahuman-like glycoprotein in a non-human host cell comprising the step ofintroducing into the cell one or more nucleic acid molecules whichencode an enzyme or enzymes for production of the Man₅GlcNAc₂carbohydrate structure. In one preferred embodiment, a nucleic acidmolecule encoding one or more mannosidase activities involved in theproduction of Man₅GlcNAc₂ from Man₈GlcNAc₂ or Man₉GlcNAc₂ is introducedinto the host. The invention additionally relates to methods for makingaltered glycoproteins in a host cell comprising the step of introducinginto the host cell a nucleic acid molecule which encodes one or moreglycosylation enzymes or activities. Preferred enzyme activities areselected from the group consisting of UDP-GlcNAc transferase,UDP-galactosyltransferase, GDP-fucosyltransferase,CMP-sialyltransferase, UDP-GlcNAc transporter, UDP-galactosetransporter, GDP-fucose transporter, CMP-sialic acid transporter, andnucleotide diphosphatases. In a particularly preferred embodiment, thehost is selected or engineered to express two or more enzymaticactivities in which the product of one activity increases substratelevels of another activity, e.g., a glycosyltransferase and acorresponding sugar transporter, e.g., GnTI and UDP-GlcNAc transporteractivities. In another preferred embodiment, the host is selected orengineered to expresses an activity to remove products which may inhibitsubsequent glycosylation reactions, e.g. a UDP- or GDP-specificdiphosphatase activity.

Preferred methods of the invention involve expressing one or moreenzymatic activities from a nucleic acid molecule in a host cell andcomprise the step of targeting at least one enzymatic activity to adesired subcellular location (e.g., an organelle) by forming a fusionprotein comprising a catalytic domain of the enzyme and a cellulartargeting signal peptide, e.g., a heterologous signal peptide which isnot normally ligated to or associated with the catalytic domain. Thefusion protein is encoded by at least one genetic construct (“fusionconstruct”) comprising a nucleic acid fragment encoding a cellulartargeting signal peptide ligated in the same translational reading frame(“in-frame”) to a nucleic acid fragment encoding an enzyme (e.g.,glycosylation enzyme), or catalytically active fragment thereof.

The targeting signal peptide component of the fusion construct orprotein is preferably derived from a member of the group consisting of:membrane-bound proteins of the ER or Golgi, retrieval signals, Type IImembrane proteins, Type I membrane proteins, membrane spanningnucleotide sugar transporters, mannosidases, sialyltransferases,glucosidases, mannosyltransferases and phosphomannosyltransferases.

The catalytic domain component of the fusion construct or protein ispreferably derived from a glycosidase, mannosidase or aglycosyltransferase activity derived from a member of the groupconsisting of GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI, GalT,Fucosyltransferase and Sialyltransferase. The catalytic domainpreferably has a pH optimum within 1.4 pH units of the average pHoptimum of other representative enzymes in the organelle in which theenzyme is localized, or has optimal activity at a pH between 5.1 and8.0. In a preferred embodiment, the catalytic domain encodes amannosidase selected from the group consisting of C. elegans mannosidaseIA, C. elegans mannosidase IB, D. melanogaster mannosidase IA, H.sapiens mannosidase IB, P. citrinum mannosidase I, mouse mannosidase IA,mouse mannosidase IB, A. nidulans mannosidase IA, A. nidulansmannosidase IB, A. nidulans mannosidase IC, mouse mannosidase II, C.elegans mannosidase II, H. sapiens mannosidase II, mannosidase Iix, andmannosidase III.

Selecting a Glycosylation Enzyme: pH Optima and Subcellular Localization

In one embodiment of the invention, a human-like glycoprotein is madeefficiently in a non-human eukaryotic host cell by introducing into asubcellular compartment of the cell a glycosylation enzyme selected tohave a pH optimum similar to the pH optima of other enzymes in thetargeted subcellular compartment. For example, most enzymes that areactive in the ER and Golgi apparatus of S. cerevisiae have pH optimathat are between about 6.5 and 7.5 (see Table 3). Because theglycosylation of proteins is a highly evolved and efficient process, theinternal pH of the ER and the Golgi is likely also in the range of about6-8. All previous approaches to reduce mannosylation by the action ofrecombinant mannosidases in fungal hosts, however, have introducedenzymes that have a pH optimum of around pH 5.0 (Martinet et al. (1998)Biotech. Letters 20(12): 1171-1177, and Chiba et al. (1998) J. Biol.Chem. 273(41): 26298-26304). At pH 7.0, the in vitro determined activityof those mannosidases is reduced to less than 10%, which is likelyinsufficient activity at their point of use, namely, the ER and earlyGolgi, for the efficient in vivo production of Man₅GlcNAc₂ on N-glycans.

Accordingly, a preferred embodiment of this invention targets a selectedglycosylation enzyme (or catalytic domain thereof), e.g., anα-mannosidase, to a subcellular location in the host cell (e.g., anorganelle) where the pH optimum of the enzyme or domain is within 1.4 pHunits of the average pH optimum of other representative marker enzymeslocalized in the same organelle(s). The pH optimum of the enzyme to betargeted to a specific organelle should be matched with the pH optimumof other enzymes found in the same organelle to maximize the activityper unit enzyme obtained. Table 3 summarizes the activity ofmannosidases from various sources and their respective pH optima. Table4 summarizes their typical subcellular locations.

TABLE 3 Mannosidases and their pH optimum. pH Source Enzyme optimumReference Aspergillus saitoi α-1,2-mannosidase 5.0 Ichishima et al.(1999) Biochem. J. 339(Pt 3): 589-597 Trichoderma reeseiα-1,2-mannosidase 5.0 Maras et al. (2000) J. Biotechnol. 77(2-3):255-263 Penicillium α-D-1,2- 5.0 Yoshida et al. (1993) citrinummannosidase Biochem. J. 290(Pt 2): 349-354 C. elegans α-1,2-mannosidase5.5 see FIG. 11 Aspergillus α-1,2-mannosidase 6.0 Eades and Hintz (2000)nidulans Gene 255(1): 25-34 Homo sapiens α-1,2-mannosidase 6.0 IA(Golgi)Homo sapiens IB α-1,2-mannosidase 6.0 (Golgi) Lepidopteran Type Iα-1,2-Man₆- 6.0 Ren et al. (1995) insect cells mannosidase Biochem.34(8): 2489-2495 Homo sapiens α-D-mannosidase 6.0 Chandrasekaran et al.(1984) Cancer Res. 44(9): 4059-68 Xanthomonas α-1,2,3-mannosidase 6.0U.S. Pat. No. 6,300,113 manihotis Mouse IB (Golgi) α-1,2-mannosidase 6.5Schneikert and Herscovics (1994) Glycobiology. 4(4): 445-50 Bacillus sp.α-D-1,2- 7.0 Maruyama et al. (1994) (secreted) mannosidase CarbohydrateRes. 251: 89-98

In a preferred embodiment, a particular enzyme or catalytic domain istargeted to a subcellular location in the host cell by means of achimeric fusion construct encoding a protein comprising a cellulartargeting signal peptide not normally associated with the enzymaticdomain. Preferably, an enzyme or domain is targeted to the ER, theearly, medial or late Golgi, or the trans Golgi apparatus of the hostcell.

In a more preferred embodiment, the targeted glycosylation enzyme is amannosidase, glycosyltransferase or a glycosidase. In an especiallypreferred embodiment, mannosidase activity is targeted to the ER or cisGolgi, where the early reactions of glycosylation occur. While thismethod is useful for producing a human-like glycoprotein in a non-humanhost cell, it will be appreciated that the method is also useful moregenerally for modifying carbohydrate profiles of a glycoprotein in anyeukaryotic host cell, including human host cells.

Targeting sequences which mediate retention of proteins in certainorganelles of the host cell secretory pathway are well-known anddescribed in the scientific literature and public databases, asdiscussed in more detail below with respect to libraries for selectionof targeting sequences and targeted enzymes. Such subcellular targetingsequences may be used alone or in combination to target a selectedglycosylation enzyme (or catalytic domain thereof) to a particularsubcellular location in a host cell, i.e., especially to one where theenzyme will have enhanced or optimal activity based on pH optima or thepresence of other stimulatory factors.

When one attempts to trim high mannose structures to yield Man₅GlcNAc₂in the ER or the Golgi apparatus of a host cell such as S. cerevisiae,for example, one may choose any enzyme or combination of enzymes that(1) has a sufficiently close pH optimum (i.e., between pH 5.2 and pH7.8), and (2) is known to generate, alone or in concert, the specificisomeric Man₅GlcNAc₂ structure required to accept subsequent addition ofGlcNAc by GnTI. Any enzyme or combination of enzymes that is shown togenerate a structure that can be converted to GlcNAcMan₅GlcNAc₂ by GnTIin vitro would constitute an appropriate choice. This knowledge may beobtained from the scientific literature or experimentally. For example,one may determine whether a potential mannosidase can convertMan₈GlcNAc₂-2AB (2-aminobenzamide) to Man₅GlcNAc₂-AB and then verifythat the obtained Man₅GlcNAc₂-2AB structure can serve a substrate forGnTI and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro. Mannosidase IAfrom a human or murine source, for example, would be an appropriatechoice (see, e.g., Example 4). Examples described herein utilize2-aminobenzamide labeled N-linked oligomannose followed by HPLC analysisto make this determination.

TABLE 4 Cellular location and pH optima of various glycosylation-relatedenzymes of S. cerevisiae. Lo- pH op- Gene Activity cation timumReference(s) KTR1 α-1,2 Golgi 7.0 Romero et al. mannosyltransferase(1997) Biochem. J. 321(Pt 2): 289-295 MNS1 α-1,2-mannosidase ER 6.5Lipari et al. (1994) Glycobiology. Oct; 4(5): 697-702 CWH41 glucosidaseI ER 6.8 — mannosyltransferase Golgi 7-8 Lehele and Tanner (1974)Biochim. Biophys. Acta 350(1): 225-235 KRE2 α-1,2 Golgi 6.5-9.0 Romeroet al. mannosyltransferase (1997) Biochem. J. 321(Pt 2): 289-295

Accordingly, a glycosylation enzyme such as an α-1,2-mannosidase enzymeused according to the invention has an optimal activity at a pH ofbetween 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH of between 5.5 and 7.5. The C. elegansmannosidase enzyme, for example, works well in the methods of theinvention and has an apparent pH optimum of about 5.5). Preferredmannosidases include those listed in Table 3 having appropriate pHoptima, e.g. Aspergillus nidulans, Homo sapiens IA (Golgi), Homo sapiensIB (Golgi), Lepidopteran insect cells (IPLB-SF21AE), Homo sapiens, mouseIB (Golgi), Xanthomonas manihotis, Drosophila melanogaster and C.elegans.

An experiment which illustrates the pH optimum for an α-1,2-mannosidaseenzyme is described in Example 7. A chimeric fusion protein BB27-2(Saccharomyces MNN10(s)/C. elegans mannosidase IB Δ31), which leaks intothe medium was subjected to various pH ranges to determine the optimalactivity of the enzyme. The results of the experiment show that theα-1,2-mannosidase has an optimal pH of about 5.5 for its function (FIG.11).

In a preferred embodiment, a single cloned mannosidase gene is expressedin the host organism. However, in some cases it may be desirable toexpress several different mannosidase genes, or several copies of oneparticular gene, in order to achieve adequate production of Man₅GlcNAc₂.In cases where multiple genes are used, the encoded mannosidasespreferably all have pH optima within the preferred range of about 5.1 toabout 8.0, or especially between about 5.5 and about 7.5. Preferredmannosidase activities include α-1,2-mannosidases derived from mouse,human, Lepidoptera, Aspergillus nidulans, or Bacillus sp., C. elegans,D. melanogaster, P. citrinum, X. laevis or A. nidulans.

In Vivo Alteration of Host Cell Glycosylation Using a Combinatorial DNALibrary

Certain methods of the invention are preferably (but need notnecessarily be) carried out using one or more nucleic acid libraries. Anexemplary feature of a combinatorial nucleic acid library of theinvention is that it comprises sequences encoding cellular targetingsignal peptides and sequences encoding proteins to be targeted (e.g.,enzymes or catalytic domains thereof, including but not limited to thosewhich mediate glycosylation).

In one embodiment, a combinatorial nucleic acid library comprises: (a)at least two nucleic acid sequences encoding different cellulartargeting signal peptides; and (b) at least one nucleic acid sequenceencoding a polypeptide to be targeted. In another embodiment, acombinatorial nucleic acid library comprises: (a) at least one nucleicacid sequence encoding a cellular targeting signal peptide; and (b) atleast two nucleic acid sequences encoding a polypeptide to be targetedinto a host cell. As described further below, a nucleic acid sequencederived from (a) and a nucleic acid sequence derived from (b) areligated to produce one or more fusion constructs encoding a cellulartargeting signal peptide functionally linked to a polypeptide domain ofinterest. One example of a functional linkage is when the cellulartargeting signal peptide is ligated to the polypeptide domain ofinterest in the same translational reading frame (“in-frame”).

In a preferred embodiment, a combinatorial DNA library expresses one ormore fusion proteins comprising cellular targeting signal peptidesligated in-frame to catalytic enzyme domains. The encoded fusion proteinpreferably comprises a catalytic domain of an enzyme involved inmammalian- or human-like modification of N-glycans. In a more preferredembodiment, the catalytic domain is derived from an enzyme selected fromthe group consisting of mannosidases, glycosyltransferases and otherglycosidases which is ligated in-frame to one or more targeting signalpeptides. The enzyme domain may be exogenous and/or endogenous to thehost cell. A particularly preferred signal peptide is one normallyassociated with a protein that undergoes ER to Golgi transport.

The combinatorial DNA library of the present invention may be used forproducing and localizing in vivo enzymes involved in mammalian- orhuman-like N-glycan modification. The fusion constructs of thecombinatorial DNA library are engineered so that the encoded enzymes arelocalized in the ER, Golgi or the trans-Golgi network of the host cellwhere they are involved in producing particular N-glycans on aglycoprotein of interest. Localization of N-glycan modifying enzymes ofthe present invention is achieved through an anchoring mechanism orthrough protein-protein interaction where the localization peptideconstructed from the combinatorial DNA library localizes to a desiredorganelle of the secretory pathway such as the ER, Golgi or the transGolgi network.

An example of a useful N-glycan, which is produced efficiently and insufficient quantities for further modification by human-like (complex)glycosylation reactions is Man₅GlcNAc₂. A sufficient amount ofMan₅GlcNAc₂ is needed on a glycoprotein of interest for furtherhuman-like processing in vivo (e.g., more than 30 mole %). TheMan₅GlcNAc₂ intermediate may be used as a substrate for further N-glycanmodification to produce GlcNAcMan₅GlcNAc₂ (FIG. 1B; see above).Accordingly, the combinatorial DNA library of the present invention maybe used to produce enzymes that subsequently produce GlcNAcMan₅GlcNAc₂,or other desired complex N-glycans, in a useful quantity.

A further aspect of the fusion constructs produced using thecombinatorial DNA library of the present invention is that they enablesufficient and often near complete intracellular N-glycan trimmingactivity in the engineered host cell. Preferred fusion constructsproduced by the combinatorial DNA library of the invention encode aglycosylation enzyme, e.g., a mannosidase, which is effectivelylocalized to an intracellular host cell compartment and thereby exhibitsvery little and preferably no extracellular activity. The preferredfusion constructs of the present invention that encode a mannosidaseenzyme are shown to localize where the N-glycans are modified, namely,the ER and the Golgi. The fusion enzymes of the present invention aretargeted to such particular organelles in the secretory pathway wherethey localize and act upon N-glycans such as Man₈GlcNAc₂ to produceMan₅GlcNAc₂ on a glycoprotein of interest.

GnTIII fusion constructs generated from a combinatorial DNA library toproduce bisected glycans were assayed to determine any extracellularactivity: An example of a GnTIII fusion constructs exhibiting in vivoalteration of host cell glycosylation is designated pVA53. Aftertransforming P. pastoris YSH-1 with the fusion construct pVA53, thesupernatant was tested to detect any ex vivo GnTIII activity. FIG. 33shows no apparent change in the standard substrate GlcNAcMan₅GlcNAc₂under conditions that would reveal extracellular GnTIII activity in themedium (Example 22). Similarly, FIG. 34 shows no detectableextracellular GnTIII activity in the medium in P. pastoris YSH-57reacting with the substrate GlcNAc₂Man₃GlcNAc₂ (Example 23).

Enzymes produced by the combinatorial DNA library of the presentinvention can modify N-glycans on a glycoprotein of interest as shownfor K3 or IFN-β proteins expressed in P. pastoris, as shown in FIGS. 5,6, and 25-34 (see also Examples 2, 4, and 18-23). It is, however,appreciated that other types of glycoproteins, without limitation,including erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgG fragments, IgM, urokinase, chymase, ureatrypsin inhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, osteoprotegerin, α-1 antitrypsin, DNase II, and α-fetoproteins may be glycosylated in this way.

Constructing a Combinatorial DNA Library of Fusion Constructs:

A combinatorial DNA library of fusion constructs features one or morecellular targeting signal peptides (“targeting peptides”) generallyderived from N-terminal domains of native proteins (e.g., by makingC-terminal deletions). Some targeting peptides, however, are derivedfrom the C-terminus of native proteins (e.g. SEC12). Membrane-boundproteins of the ER or the Golgi are preferably used as a source fortargeting peptide sequences. These proteins have sequences encoding acytosolic tail (ct), a transmembrane domain (tmd) and a stem region (sr)which are varied in length. These regions are recognizable by proteinsequence alignments and comparisons with known homologs and/or otherlocalized proteins (e.g., comparing hydrophobicity plots).

The targeting peptides are indicated herein as short (s), medium (m) andlong (l) relative to the parts of a type II membrane. The targetingpeptide sequence indicated as short (s) corresponds to the transmembranedomain (tmd) of the membrane-bound protein. The targeting peptidesequence indicated as long (l) corresponds to the length of thetransmembrane domain (tmd) and the stem region (sr). The targetingpeptide sequence indicated as medium (m) corresponds to thetransmembrane domain (tmd) and approximately half the length of the stemregion (sr). The catalytic domain regions are indicated herein by thenumber of nucleotide deletion with respect to its wild-typeglycosylation enzyme.

Sub-Libraries

In some cases a combinatorial nucleic acid library of the invention maybe assembled directly from existing or wild-type genes. In a preferredembodiment, the DNA library is assembled from the fusion of two or moresub-libraries. By the in-frame ligation of the sub-libraries, it ispossible to create a large number of novel genetic constructs encodinguseful targeted protein domains such as those which have glycosylationactivities.

Catalytic Domain Sub-Libraries Encoding Glycosylation Activities

One useful sub-library includes DNA sequences encoding enzymes such asglycosidases (e.g., mannosidases), glycosyltransferases (e.g.,fucosyl-transferases, galactosyltransferases, glucosyltransferases),GlcNAc transferases and sialyltransferases. Catalytic domains may beselected from the host to be engineered, as well as from other relatedor unrelated organisms. Mammalian, plant, insect, reptile, algal orfungal enzymes are all useful and should be chosen to represent a broadspectrum of biochemical properties with respect to temperature and pHoptima. In a preferred embodiment, genes are truncated to give fragmentssome of which encode the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci.

The choice of such catalytic domains may be guided by the knowledge ofthe particular environment in which the catalytic domain is subsequentlyto be active. For example, if a particular glycosylation enzyme is to beactive in the late Golgi, and all known enzymes of the host organism inthe late Golgi have a certain pH optimum, or the late Golgi is known tohave a particular pH, then a catalytic domain is chosen which exhibitsadequate, and preferably maximum, activity at that pH, as discussedabove.

Targeting Peptide Sequence Sub-Libraries

Another useful sub-library includes nucleic acid sequences encodingtargeting signal peptides that result in localization of a protein to aparticular location within the ER, Golgi, or trans Golgi network. Thesetargeting peptides may be selected from the host organism to beengineered as well as from other related or unrelated organisms.Generally such sequences fall into three categories: (1) N-terminalsequences encoding a cytosolic tail (ct), a transmembrane domain (tmd)and part or all of a stem region (sr), which together or individuallyanchor proteins to the inner (lumenal) membrane of the Golgi; (2)retrieval signals which are generally found at the C-terminus such asthe HDEL (SEQ ID NO:41) or KDEL (SEQ ID NO:42) tetrapeptide; and (3)membrane spanning regions from various proteins, e.g., nucleotide sugartransporters, which are known to localize in the Golgi.

In the first case, where the targeting peptide consists of variouselements (ct, tmd and sr), the library is designed such that the ct, thetmd and various parts of the stem region are represented. Accordingly, apreferred embodiment of the sub-library of targeting peptide sequencesincludes ct, tmd, and/or sr sequences from membrane-bound proteins ofthe ER or Golgi. In some cases it may be desirable to provide thesub-library with varying lengths of sr sequence. This may beaccomplished by PCR using primers that bind to the 5′ end of the DNAencoding the cytosolic region and employing a series of opposing primersthat bind to various parts of the stem region.

Still other useful sources of targeting peptide sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL or KDEL, whichare typically found at the C-terminus of proteins that are transportedretrograde into the ER or Golgi. Still other sources of targetingpeptide sequences include (a) type II membrane proteins, (b) the enzymeslisted in Table 3, (c) membrane spanning nucleotide sugar transportersthat are localized in the Golgi, and (d) sequences referenced in Table5.

TABLE 5 Sources of useful compartmental targeting sequences Gene orLocation of Sequence Organism Function Gene Product MNSI A. nidulansα-1,2-mannosidase ER MNSI A. niger α-1,2-mannosidase ER MNSI S.cerevisiae α-1,2-mannosidase ER GLSI S. cerevisiae glucosidase ER GLSIA. niger glucosidase ER GLSI A. nidulans glucosidase ER HDEL Universalin retrieval signal ER at C- fungi terminus SEC12 S. cerevisiae COPIIvesicle protein ER/Golgi SEC12 A. niger COPII vesicle protein ER/GolgiOCH1 S. cerevisiae 1,6-mannosyltransferase Golgi (cis) OCH1 P. pastoris1,6-mannosyltransferase Golgi (cis) MNN9 S. cerevisiae1,6-mannosyltransferase Golgi complex MNN9 A. niger undetermined GolgiVAN1 S. cerevisiae undetermined Golgi VAN1 A. niger undetermined GolgiANP1 S. cerevisiae undetermined Golgi HOCI S. cerevisiae undeterminedGolgi MNN10 S. cerevisiae undetermined Golgi MNN10 A. niger undeterminedGolgi MNN11 S. cerevisiae undetermined Golgi (cis) MNN11 A. nigerundetermined Golgi (cis) MNT1 S. cerevisiae 1,2-mannosyltransferaseGolgi (cis, medial) KTR1 P. pastoris undetermined Golgi (medial) KRE2 P.pastoris undetermined Golgi (medial) KTR3 P. pastoris undetermined Golgi(medial) MNN2 S. cerevisiae 1,2-mannosyltransferase Golgi (medial) KTR1S. cerevisiae undetermined Golgi (medial) KTR2 S. cerevisiaeundetermined Golgi (medial) MNN1 S. cerevisiae 1,3-mannosyltransferaseGolgi (trans) MNN6 S. cerevisiae Phosphomannosyltransferase Golgi(trans) 2,6 ST H. sapiens 2,6-sialyltransferase trans Golgi network UDP-S. pombe UDP-Gal transporter Golgi Gal T

In any case, it is highly preferred that targeting peptide sequences areselected which are appropriate for the particular enzymatic activity oractivities to function optimally within the sequence of desiredglycosylation reactions. For example, in developing a modifiedmicroorganism capable of terminal sialylation of nascent N-glycans, aprocess which occurs in the late Golgi in humans, it is desirable toutilize a sub-library of targeting peptide sequences derived from lateGolgi proteins. Similarly, the trimming of Man₈GlcNAc₂ by anα-1,2-mannosidase to give Man₅GlcNAc₂ is an early step in complexN-glycan formation in humans (FIG. 1B). It is therefore desirable tohave this reaction occur in the ER or early Golgi of an engineered hostmicroorganism. A sub-library encoding ER and early Golgi retentionsignals is used.

A series of fusion protein constructs (i.e., a combinatorial DNAlibrary) is then constructed by functionally linking one or a series oftargeting peptide sequences to one or a series of sequences encodingcatalytic domains. In a preferred embodiment, this is accomplished bythe in-frame ligation of a sub-library comprising DNA encoding targetingpeptide sequences (above) with a sub-library comprising DNA encodingglycosylation enzymes or catalytically active fragments thereof (seebelow).

The resulting library comprises synthetic genes encoding targetingpeptide sequence-containing fusion proteins. In some cases it isdesirable to provide a targeting peptide sequence at the N-terminus of afusion protein, or in other cases at the C-terminus. In some cases,targeting peptide sequences may be inserted within the open readingframe of an enzyme, provided the protein structure of individual foldeddomains is not disrupted. Each type of fusion protein is constructed (ina step-wise directed or semi-random fashion) and optimal constructs maybe selected upon transformation of host cells and characterization ofglycosylation patterns in transformed cells using methods of theinvention.

Alteration of Host Cell Glycosylation Using Fusion Constructs FromCombinatorial Libraries:

The construction of a preferred combinatorial DNA library is illustratedschematically in FIG. 2 and described in Example 4. The fusion constructmay be operably linked to a multitude of vectors, such as expressionvectors well-known in the art. A wide variety of such fusion constructswere assembled using representative activities as shown in Table 6.Combinations of targeting peptide/catalytic domains may be assembled foruse in targeting mannosidase, glycosyltransferase and glycosidaseactivities in the ER, Golgi, and the trans Golgi network according tothe invention. Surprisingly, the same catalytic domain may have noeffect to a very profound effect on N-glycosylation patterns, dependingon the type of targeting peptide used (see, e.g., Table 7, Example 4).

Mannosidase Fusion Constructs

A representative example of a mannosidase fusion construct derived froma combinatorial DNA library of the invention is pFB8, which a truncatedSaccharomyces SEC12(m) targeting peptide (988-1296 nucleotides of SEC12from SwissProt P11655) ligated in-frame to a 187 N-terminal amino aciddeletion of a mouse α-mannosidase IA (Genbank AN 6678787). Thenomenclature used herein, thus, refers to the targetingpeptide/catalytic domain region of a glycosylation enzyme asSaccharomyces SEC12(m)/mouse mannosidase IA Δ187. The encoded fusionprotein localizes in the ER by means of the SEC12 targeting peptidesequence while retaining its mannosidase catalytic domain activity andis capable of producing in vivo N-glycans having a Man₅GlcNAc₂ structure(Example 4; FIGS. 6F and 7B).

The fusion construct pGC5, Saccharomyces MNS1(m)/mouse mannosidase IBΔ99, is another example of a fusion construct having intracellularmannosidase trimming activity (Example 4; FIGS. 5D and 8B). Fusionconstruct pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)is yet another example of an efficient fusion construct capable ofproducing N-glycans having a Man₅GlcNAc₂ structure in vivo. By creatinga combinatorial DNA library of these and other such mannosidase fusionconstructs according to the invention, a skilled artisan may distinguishand select those constructs having optimal intracellular trimmingactivity from those having relatively low or no activity. Methods usingcombinatorial DNA libraries of the invention are advantageous becauseonly a select few mannosidase fusion constructs may produce aparticularly desired N-glycan in vivo.

In addition, mannosidase trimming activity may be specific to aparticular protein of interest. Thus, it is to be further understoodthat not all targeting peptide/mannosidase catalytic domain fusionconstructs may function equally well to produce the proper glycosylationon a glycoprotein of interest. Accordingly, a protein of interest may beintroduced into a host cell transfected with a combinatorial DNA libraryto identify one or more fusion constructs which express a mannosidaseactivity optimal for the protein of interest. One skilled in the artwill be able to produce and select optimal fusion construct(s) using thecombinatorial DNA library approach described herein.

It is apparent, moreover, that other such fusion constructs exhibitinglocalized active mannosidase catalytic domains (or more generally,domains of any enzyme) may be made using techniques such as thoseexemplified in Example 4 and described herein. It will be a matter ofroutine experimentation for one skilled in the art to make and use thecombinatorial DNA library of the present invention to optimize, forexample, Man₅GlcNAc₂ production from a library of fusion constructs in aparticular expression vector introduced into a particular host cell.

Glycosyltransferase Fusion Constructs

Similarly, a glycosyltransferase combinatorial DNA library was madeusing the methods of the invention. A combinatorial DNA library ofsequences derived from glycosyltransferase I (GnTI) activities wereassembled with targeting peptides and screened for efficient productionin a lower eukaryotic host cell of a GlcNAcMan₅GlcNAc₂ N-glycanstructure on a marker glycoprotein. A fusion construct shown to produceGlcNAcMan₅GlcNAc₂ (pPB104), Saccharomyces MNN9(s)/human GnTI Δ38 wasidentified (Example 8). A wide variety of such GnTI fusion constructswere assembled (Example 8, Table 10). Other combinations of targetingpeptide/GnTI catalytic domains can readily be assembled by making acombinatorial DNA library. It is also apparent to one skilled in the artthat other such fusion constructs exhibiting glycosyltransferaseactivity may be made as demonstrated in Example 8. It will be a matterof routine experimentation for one skilled in the art to use thecombinatorial DNA library method described herein to optimizeGlcNAcMan₅GlcNAc₂ production using a selected fusion construct in aparticular expression vector and host cell line.

As stated above for mannosidase fusion constructs, not all targetingpeptide/GnTI catalytic domain fusion constructs will function equallywell to produce the proper glycosylation on a glycoprotein of interestas described herein. However, one skilled in the art will be able toproduce and select optimal fusion construct(s) using a DNA libraryapproach as described herein. Example 8 illustrates a preferredembodiment of a combinatorial DNA library comprising targeting peptidesand GnTI catalytic domain fusion constructs involved in producingglycoproteins with predominantly GlcNAcMan₅GlcNAc₂ structure.

Using Multiple Fusion Constructs to Alter Host Cell Glycosylation

In another example of using the methods and libraries of the inventionto alter host cell glycosylation, a P. pastoris strain with an OCH1deletion that expresses a reporter protein (K3) was transformed withmultiple fusion constructs isolated from combinatorial libraries of theinvention to convert high mannose N-glycans to human-like N-glycans(Example 8). First, the mannosidase fusion construct pFB8 (SaccharomycesSEC12(m)/mouse mannosidase IA Δ187) was transformed into a P. pastorisstrain lacking 1,6 initiating mannosyltransferases activity (i.e., och1deletion; Example 1). Second, pPB103 comprising a K. lactis MNN2-2 gene(Genbank AN AF 106080) encoding an UDP-GlcNAc transporter wasconstructed to increase further production of GlcNAcMan₅GlcNAc₂. Theaddition of the UDP-GlcNAc transporter increased production ofGlcNAcMan₅GlcNAc₂ significantly in the P. pastoris strain as illustratedin FIG. 10B. Third, pPB104 comprising Saccharomyces MNN9(s)/human GnTIΔ38 was introduced into the strain. This P. pastoris strain is referredto as “PBP-3.” (See FIG. 36.)

It is understood by one skilled in the art that host cells such as theabove-described yeast strains can be sequentially transformed and/orco-transformed with one or more expression vectors. It is alsounderstood that the order of transformation is not particularly relevantin producing the glycoprotein of interest. The skilled artisanrecognizes the routine modifications of the procedures disclosed hereinmay provide improved results in the production of the glycoprotein ofinterest.

The importance of using a particular targeting peptide sequence with aparticular catalytic domain sequence becomes readily apparent from theexperiments described herein. The combinatorial DNA library provides atool for constructing enzyme fusions that are involved in modifyingN-glycans on a glycoprotein of interest, which is especially useful inproducing human-like glycoproteins. (Any enzyme fusion, however, may beselected using libraries and methods of the invention.) Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce K3 with N-glycans of the structure Man₅GlcNAc₂as shown in FIGS. 5D and 5E. This confers a reduced molecular mass tothe cleaved glycan compared to the K3 of the parent OCH1 deletionstrain, as was detected by MALDI-TOF mass spectrometry in FIG. 5C.

Similarly, the same approach was used to produce another secretedglycoprotein: IFN-β comprising predominantly Man₅GlcNAc₂. TheMan₅GlcNAc₂ was removed by PNGase digestion (Papac et al. (1998)Glycobiology 8:445-454) and subjected to MALDI-TOF as shown in FIGS.6A-6F. A single prominent peak at 1254 (m/z) confirms Man₅GlcNA₂production on IFN-β in FIG. 6E (pGC5) (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) and 6F (pFB8) (Saccharomyces SEC12(m)/mousemannosidase IA Δ187). Furthermore, in the P. pastoris strain PBP-3comprising pFB8 (Saccharomyces SEC12(m)/mouse mannosidase IA Δ187),pPB104 (Saccharomyces MNN9 (s)/human GnTI Δ38) and pPB103 (K. lactisMNN2-2 gene), the hybrid N-glycan GlcNAcMan₅GlcNAc₂ [b] was detected byMALDI-TOF (FIG. 10).

After identifying transformants with a high degree of mannose trimming,additional experiments were performed to confirm that mannosidase(trimming) activity occurred in vivo and was not predominantly theresult of extracellular activity in the growth medium (Example 6; FIGS.7-9).

Although the present invention is exemplified using a P. pastoris hostorganism, it is understood by those skilled in the art that othereukaryotic host cells, including other species of yeast and fungalhosts, may be altered as described herein to produce human-likeglycoproteins. The techniques described herein for identification anddisruption of undesirable host cell glycosylation genes, e.g. OCH1, isunderstood to be applicable for these and/or other homologous orfunctionally related genes in other eukaryotic host cells such as otheryeast and fungal strains. As described in Example 9, och1 mnn1 geneswere deleted from K. lactis to engineer a host cell leading to N-glycansthat are completely converted to Man₅GlcNAc₂ by 1,2-mannosidase (FIG.12C).

The MNN1 gene was cloned from K. lactis as described in Example 9. Usinggene-specific primers, a construct was made to delete the MNN1 gene fromthe genome of K. lactis (Example 9). Host cells depleted in och1 andmnn1 activities produce N-glycans having a Man₉GlcNAc₂ carbohydratestructure (see, e.g., FIG. 12B). Such host cells may be engineeredfurther using, e.g., methods and libraries of the invention, to producemammalian- or human-like glycoproteins.

Thus, in another embodiment, the invention provides an isolated nucleicacid molecule having a nucleic acid sequence comprising or consisting ofat least forty-five, preferably at least 50, more preferably at least 60and most preferably 75 or more nucleotide residues of the K. lactis MNN1gene, and homologs, variants and derivatives thereof. The invention alsoprovides nucleic acid molecules that hybridize under stringentconditions to the above-described nucleic acid molecules. Similarly,isolated polypeptides (including muteins, allelic variants, fragments,derivatives, and analogs) encoded by the nucleic acid molecules of theinvention are provided. In addition, also provided are vectors,including expression vectors, which comprise a nucleic acid molecule ofthe invention, as described further herein. Similarly host cellstransformed with the nucleic acid molecules or vectors of the inventionare provided.

Another aspect of the present invention thus relates to a non-humaneukaryotic host strain expressing glycoproteins comprising modifiedN-glycans that resemble those made by human-cells. Performing themethods of the invention in species other than yeast and fungal cells isthus contemplated and encompassed by this invention. It is contemplatedthat a combinatorial nucleic acid library of the present invention maybe used to select constructs that modify the glycosylation pathway inany eukaryotic host cell system. For example, the combinatoriallibraries of the invention may also be used in plants, algae andinsects, and in other eukaryotic host cells, including mammalian andhuman cells, to localize proteins, including glycosylation enzymes orcatalytic domains thereof, in a desired location along a host cellsecretory pathway. Preferably, glycosylation enzymes or catalyticdomains and the like are targeted to a subcellular location along thehost cell secretory pathway where they are capable of functioning, andpreferably, where they are designed or selected to function mostefficiently.

Plant and insect cells may also be engineered to alter the glycosylationof expressed proteins using the combinatorial library and methods of theinvention. Furthermore, glycosylation in mammalian cells, includinghuman cells, may also be modified using the combinatorial library andmethods of the invention. It may be possible, for example, to optimize aparticular enzymatic activity or to otherwise modify the relativeproportions of various N-glycans made in a mammalian host cell using thecombinatorial library and methods of the invention.

Examples of modifications to glycosylation which can be affected using amethod according to this embodiment of the invention are: (1)engineering a eukaryotic host cell to trim mannose residues fromMan₈GlcNAc₂ to yield a Man₅GlcNAc₂ N-glycan; (2) engineering eukaryotichost cell to add an N-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂by action of GlcNAc transferase I; (3) engineering a eukaryotic hostcell to functionally express an enzyme such as an N-acetylglucosaminylTransferase (GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI), mannosidase II,fucosyltransferase (FT), galactosyl tranferase (GalT) or asialyltransferase (ST).

By repeating the method, increasingly complex glycosylation pathways canbe engineered into a target host, such as a lower eukaryoticmicroorganism. In one preferred embodiment, the host organism istransformed two or more times with DNA libraries including sequencesencoding glycosylation activities. Selection of desired phenotypes maybe performed after each round of transformation or alternatively afterseveral transformations have occurred. Complex glycosylation pathwayscan be rapidly engineered in this manner.

Sequential Glycosylation Reactions

In a preferred embodiment, such targeting peptide/catalytic domainlibraries are designed to incorporate existing information on thesequential nature of glycosylation reactions in higher eukaryotes.Reactions known to occur early in the course of glycoprotein processingrequire the targeting of enzymes that catalyze such reactions to anearly part of the Golgi or the ER. For example, the trimming ofMan₈GlcNAc₂ to Man₅GlcNAc₂ by mannosidases is an early step in complexN-glycan formation (FIGS. 1B and 35A). Because protein processing isinitiated in the ER and then proceeds through the early, medial and lateGolgi, it is desirable to have this reaction occur in the ER or earlyGolgi. When designing a library for mannosidase I localization, forexample, one thus attempts to match ER and early Golgi targeting signalswith the catalytic domain of mannosidase I.

Generating Additional Sequence Diversity

The method of this embodiment is most effective when a nucleic acid,e.g., a DNA library transformed into the host contains a large diversityof sequences, thereby increasing the probability that at least onetransformant will exhibit the desired phenotype. Single amino acidmutations, for example, may drastically alter the activity ofglycoprotein processing enzymes (Romero et al. (2000) J. Biol. Chem.275(15):11071-4). Accordingly, prior to transformation, a DNA library ora constituent sub-library may be subjected to one or more techniques togenerate additional sequence diversity. For example, one or more roundsof gene shuffling, error prone PCR, in vitro mutagenesis or othermethods for generating sequence diversity, may be performed to obtain alarger diversity of sequences within the pool of fusion constructs.

Expression Control Sequences

In addition to the open reading frame sequences described above, it isgenerally preferable to provide each library construct with expressioncontrol sequences, such as promoters, transcription terminators,enhancers, ribosome binding sites, and other functional sequences as maybe necessary to ensure effective transcription and translation of thefusion proteins upon transformation of fusion constructs into the hostorganism.

Suitable vector components, e.g., selectable markers, expression controlsequences (e.g., promoter, enhancers, terminators and the like) and,optionally, sequences required for autonomous replication in a hostcell, are selected as a function of which particular host cell ischosen. Selection criteria for suitable vector components for use in aparticular mammalian or a lower eukaryotic host cell are routine.Preferred lower eukaryotic host cells of the invention include Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp. Fusariumgramineum, Fusarium venenatum and Neurospora crassa. Where the host isPichia pastoris, suitable promoters include, for example, the AOX1,AOX2, GAPDH and P40 promoters.

Selectable Markers

It is also preferable to provide each construct with at least oneselectable marker, such as a gene to impart drug resistance or tocomplement a host metabolic lesion. The presence of the marker is usefulin the subsequent selection of transformants; for example, in yeast theURA3, HIS4, SUC2, G418, BLA, or SH BLE genes may be used. A multitude ofselectable markers are known and available for use in yeast, fungi,plant, insect, mammalian and other eukaryotic host cells.

Transformation

The nucleic acid library is then transformed into the host organism. Inyeast, any convenient method of DNA transfer may be used, such aselectroporation, the lithium chloride method, or the spheroplast method.In filamentous fungi and plant cells, conventional methods includeparticle bombardment, electroporation and agrobacterium mediatedtransformation. To produce a stable strain suitable for high-densityculture (e.g., fermentation in yeast), it is desirable to integrate theDNA library constructs into the host chromosome. In a preferredembodiment, integration occurs via homologous recombination, usingtechniques well-known in the art. For example, DNA library elements areprovided with flanking sequences homologous to sequences of the hostorganism. In this manner, integration occurs at a defined site in thehost genome, without disruption of desirable or essential genes.

In an especially preferred embodiment, library DNA is integrated intothe site of an undesired gene in a host chromosome, effecting thedisruption or deletion of the gene. For example, integration into thesites of the OCH1, MNN1, or MNN4 genes allows the expression of thedesired library DNA while preventing the expression of enzymes involvedin yeast hypermannosylation of glycoproteins. In other embodiments,library DNA may be introduced into the host via a nucleic acid molecule,plasmid, vector (e.g., viral or retroviral vector), chromosome, and maybe introduced as an autonomous nucleic acid molecule or by homologous orrandom integration into the host genome. In any case, it is generallydesirable to include with each library DNA construct at least oneselectable marker gene to allow ready selection of host organisms thathave been stably transformed. Recyclable marker genes such as ura3,which can be selected for or against, are especially suitable.

Screening and Selection Processes

After transformation of the host strain with the DNA library,transformants displaying a desired glycosylation phenotype are selected.Selection may be performed in a single step or by a series of phenotypicenrichment and/or depletion steps using any of a variety of assays ordetection methods. Phenotypic characterization may be carried outmanually or using automated high-throughput screening equipment.Commonly, a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized.

One may screen for those cells that have the highest concentration ofterminal GlcNAc on the cell surface, for example, or for those cellswhich secrete the protein with the highest terminal GlcNAc content. Sucha screen may be based on a visual method, like a staining procedure, theability to bind specific terminal GlcNAc binding antibodies or lectinsconjugated to a marker (such lectins are available from E.Y.Laboratories Inc., San Mateo, Calif.), the reduced ability of specificlectins to bind to terminal mannose residues, the ability to incorporatea radioactively labeled sugar in vitro, altered binding to dyes orcharged surfaces, or may be accomplished by using a FluorescenceAssisted Cell Sorting (FACS) device in conjunction with a fluorophorelabeled lectin or antibody (Guillen et al. (1998) Proc. Natl. Acad. Sci.USA 95(14):7888-7892).

Accordingly, intact cells may be screened for a desired glycosylationphenotype by exposing the cells to a lectin or antibody that bindsspecifically to the desired N-glycan. A wide variety ofoligosaccharide-specific lectins are available commercially (e.g., fromEY Laboratories, San Mateo, Calif.). Alternatively, antibodies tospecific human or animal N-glycans are available commercially or may beproduced using standard techniques. An appropriate lectin or antibodymay be conjugated to a reporter molecule, such as a chromophore,fluorophore, radioisotope, or an enzyme having a chromogenic substrate(Guillen et al., 1998. Proc. Natl. Acad. Sci. USA 95(14): 7888-7892).

Screening may then be performed using analytical methods such asspectrophotometry, fluorimetry, fluorescence activated cell sorting, orscintillation counting. In other cases, it may be necessary to analyzeisolated glycoproteins or N-glycans from transformed cells. Proteinisolation may be carried out by techniques known in the art. In apreferred embodiment, a reporter protein is secreted into the medium andpurified by affinity chromatography (e.g. Ni-affinity orglutathione-S-transferase affinity chromatography). In cases where anisolated N-glycan is preferred, an enzyme such asendo-β-N-acetylglucosaminidase (Genzyme Co., Boston, Mass.; New EnglandBiolabs, Beverly, Mass.) may be used to cleave the N-glycans fromglycoproteins. Isolated proteins or N-glycans may then be analyzed byliquid chromatography (e.g., HPLC), mass spectroscopy, or other suitablemeans. U.S. Pat. No. 5,595,900 teaches several methods by which cellswith desired extracellular carbohydrate structures may be identified. Ina preferred embodiment, MALDI-TOF mass spectrometry is used to analyzethe cleaved N-glycans.

Prior to selection of a desired transformant, it may be desirable todeplete the transformed population of cells having undesired phenotypes.For example, when the method is used to engineer a functionalmannosidase activity into cells, the desired transformants will havelower levels of mannose in cellular glycoprotein. Exposing thetransformed population to a lethal radioisotope of mannose in the mediumdepletes the population of transformants having the undesired phenotype,i.e., high levels of incorporated mannose (Huffaker and Robbins (1983)Proc Natl Acad Sci USA. 80(24):7466-70). Alternatively, a cytotoxiclectin or antibody, directed against an undesirable N-glycan, may beused to deplete a transformed population of undesired phenotypes (e.g.,Stanley and Siminovitch (1977) Somatic Cell Genet 3(4):391-405). U.S.Pat. No. 5,595,900 teaches several methods by which cells with a desiredextracellular carbohydrate structures may be identified. Repeatedlycarrying out this strategy allows for the sequential engineering of moreand more complex glycans in lower eukaryotes.

To detect host cells having on their surface a high degree of thehuman-like N-glycan intermediate GlcNAcMan₃GlcNAc₂, for example, one mayselect for transformants that allow for the most efficient transfer ofGlcNAc by GlcNAc Transferase from UDP-GlcNAc in an in vitro cell assay.This screen may be carried out by growing cells harboring thetransformed library under selective pressure on an agar plate andtransferring individual colonies into a 96-well microtiter plate. Aftergrowing the cells, the cells are centrifuged, the cells resuspended inbuffer, and after addition of UDP-GlcNAc and GnTII, the release of UDPis determined either by HPLC or an enzyme linked assay for UDP.Alternatively, one may use radioactively labeled UDP-GlcNAc and GnTII,wash the cells and then look for the release of radioactive GlcNAc byN-actylglucosaminidase. All this may be carried manually or automatedthrough the use of high throughput screening equipment. Transformantsthat release more UDP, in the first assay, or more radioactively labeledGlcNAc in the second assay, are expected to have a higher degree ofGlcNAcMan₃GlcNAc₂ on their surface and thus constitute the desiredphenotype. Similar assays may be adapted to look at the N-glycans onsecreted proteins as well.

Alternatively, one may use any other suitable screen such as a lectinbinding assay that is able to reveal altered glycosylation patterns onthe surface of transformed cells. In this case the reduced binding oflectins specific to terminal mannoses may be a suitable selection tool.Galantus nivalis lectin binds specifically to terminal α-1,3 mannose,which is expected to be reduced if sufficient mannosidase II activity ispresent in the Golgi. One may also enrich for desired transformants bycarrying out a chromatographic separation step that allows for theremoval of cells containing a high terminal mannose content. Thisseparation step would be carried out with a lectin column thatspecifically binds cells with a high terminal mannose content (e.g.,Galantus nivalis lectin bound to agarose, Sigma, St. Louis, Mo.) overthose that have a low terminal mannose content.

In addition, one may directly create such fusion protein constructs, asadditional information on the localization of active carbohydratemodifying enzymes in different lower eukaryotic hosts becomes availablein the scientific literature. For example, it is known that humanβ1,4-GalTr can be fused to the membrane domain of MNT, amannosyltransferase from S. cerevisiae, and localized to the Golgiapparatus while retaining its catalytic activity (Schwientek et al.(1995) J. Biol. Chem. 270(10):5483-9). If S. cerevisiae or a relatedorganism is the host to be engineered one may directly incorporate suchfindings into the overall strategy to obtain complex N-glycans from sucha host. Several such gene fragments in P. pastoris have been identifiedthat are related to glycosyltransferases in S. cerevisiae and thus couldbe used for that purpose.

Integration Sites

As one ultimate goal of this genetic engineering effort is a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the host(e.g., fungal) chromosome preferably involves careful planning. Theengineered strain may likely have to be transformed with a range ofdifferent genes, and these genes will have to be transformed in a stablefashion to ensure that the desired activity is maintained throughout thefermentation process. As described herein, any combination of variousdesired enzyme activities may be engineered into the fungal proteinexpression host, e.g., sialyltransferases, mannosidases,fucosyltransferases, galactosyltransferases, glucosyltransferases,GlcNAc transferases, ER and Golgi specific transporters (e.g. syn andantiport transporters for UDP-galactose and other precursors), otherenzymes involved in the processing of oligosaccharides, and enzymesinvolved in the synthesis of activated oligosaccharide precursors suchas UDP-galactose, CMP-N-acetylneuraminic acid. Examples of preferredmethods for modifying glycosylation in a lower eukaryotic host cell,such as Pichia pastoris, are shown in Table 6.

TABLE 6 Some preferred embodiments for modifying glycosylation in alower eukaroytic microorganism Suitable Suitable Suitable Sources ofSuitable Transporters Desired Catalytic Localization Gene and/orStructure Activities Sequences Deletions Phosphatases Man₅GlcNAc₂ α-1,2-Mns1 (N-terminus, OCH1 none mannosidase S. cerevisiae) MNN4 (murine,Och1 (N-terminus, MNN6 human, S. cerevisiae, P. pastoris) Bacillus sp.,Ktr1 A. nidulans) Mnn9 Mnt1 (S. cerevisiae) KDEL, HDEL (C-terminus)GlcNAcMan₅GlcNAc₂ GlcNAc Och1 (N-terminus, OCH1 UDP-GlcNAc TransferaseI, S. cerevisiae, P. pastoris) MNN4 transporter (human, KTR1(N-terminus) MNN6 (human, murine, murine, rat Mnn1 (N-terminus, K.lactis) etc.) S. cerevisiae) UDPase Mnt1 (N-terminus, (human) S.cerevisiae) GDPase (N-terminus, S. cerevisiae) GlcNAcMan₃GlcNAc₂mannosidase Ktr1 OCH1 UDP-GlcNAc II Mnn1 (N-terminus, MNN4 transporterS. cerevisiae) MNN6 (human, murine, Mnt1(N-terminus, S. cerevisiae) K.lactis) Kre2/Mnt1 (S. cerevisiae) UDPase Kre2 (P. pastoris) (human) Ktr1(S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) GlcNAc₍₂₋₄₎-GlcNAc Mnn1 (N-terminus, OCH1 UDP-GlcNAc Man₃GlcNAc₂ Transferase S.cerevisiae) MNN4 transporter II, III, IV, V Mnt1 (N-terminus, MNN6(human, murine, (human, S. cerevisiae) K. lactis) murine) Kre2/Mnt1 (S.cerevisiae) UDPase Kre2 (P. pastoris) (human) Ktr1 (S. cerevisiae) Ktr1(P. pastoris) Mnn1 (S. cerevisiae) Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- β-1,4- Mnn1(N-terminus, OCH1 UDP-Galactose Man₃GlcNAc₂ Galactosyl S. cerevisiae)MNN4 transporter transferase Mnt1(N-terminus, S. cerevisiae) MNN6(human, (human) Kre2/Mnt1 (S. cerevisiae) S. pombe) Kre2 (P. pastoris)Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae) NANA₍₁₋₄₎-α-2,6- KTR1 OCH1 CMP-Sialic acid Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- SialyltransferaseMNN1 (N-terminus, MNN4 transporter Man₃GlcNAc₂ (human) S. cerevisiae)MNN6 (human) α-2,3- MNT1 (N-terminus, Sialyltransferase S. cerevisiae)Kre2/Mnt1 (S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1(P. pastoris) MNN1 (S. cerevisiae)

As any strategy to engineer the formation of complex N-glycans into ahost cell such as a lower eukaryote involves both the elimination aswell as the addition of particular glycosyltransferase activities, acomprehensive scheme will attempt to coordinate both requirements. Genesthat encode enzymes that are undesirable serve as potential integrationsites for genes that are desirable. For example, 1,6 mannosyltransferaseactivity is a hallmark of glycosylation in many known lower eukaryotes.The gene encoding alpha-1,6 mannosyltransferase (OCH1) has been clonedfrom S. cerevisiae and mutations in the gene give rise to a viablephenotype with reduced mannosylation. The gene locus encoding alpha-1,6mannosyltransferase activity therefore is a prime target for theintegration of genes encoding glycosyltransferase activity. In a similarmanner, one can choose a range of other chromosomal integration sitesthat, based on a gene disruption event in that locus, are expected to:(1) improve the cells ability to glycosylate in a more human-likefashion, (2) improve the cells ability to secrete proteins, (3) reduceproteolysis of foreign proteins and (4) improve other characteristics ofthe process that facilitate purification or the fermentation processitself.

Target Glycoproteins

The methods described herein are useful for producing glycoproteins,especially glycoproteins used therapeutically in humans. Glycoproteinshaving specific glycoforms may be especially useful, for example, in thetargeting of therapeutic proteins. For example, mannose-6-phosphate hasbeen shown to direct proteins to the lysosome, which may be essentialfor the proper function of several enzymes related to lysosomal storagedisorders such as Gaucher's, Hunter's, Hurler's, Scheie's, Fabry's andTay-Sachs disease, to mention just a few. Likewise, the addition of oneor more sialic acid residues to a glycan side chain may increase thelifetime of a therapeutic glycoprotein in vivo after administration.Accordingly, host cells (e.g., lower eukaryotic or mammalian) may begenetically engineered to increase the extent of terminal sialic acid inglycoproteins expressed in the cells. Alternatively, sialic acid may beconjugated to the protein of interest in vitro prior to administrationusing a sialic acid transferase and an appropriate substrate. Changes ingrowth medium composition may be employed in addition to the expressionof enzyme activities involved in human-like glycosylation to produceglycoproteins more closely resembling human forms (Weikert et al. (1999)Nature Biotechnology 17, 1116-1121; Werner et al. (1998)Arzneimittelforschung 48(8):870-880; Andersen and Goochee (1994) Cur.Opin. Biotechnol. 5:546-549; Yang and Butler (2000) Biotechnol.Bioengin.68(4):370-380). Specific glycan modifications to monoclonal antibodies(e.g. the addition of a bisecting GlcNAc) have been shown to improveantibody dependent cell cytotoxicity (Umana et al. (1999) Nat.Biotechnol. 17(2):176-80), which may be desirable for the production ofantibodies or other therapeutic proteins.

Therapeutic proteins are typically administered by injection, orally,pulmonary, or other means. Examples of suitable target glycoproteinswhich may be produced according to the invention include, withoutlimitation: erythropoietin, cytokines such as interferon-α,interferon-β, interferon-γ, interferon-ω, and granulocyte-CSF,coagulation factors such as factor VIII, factor IX, and human protein C,soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins,urokinase, chymase, urea trypsin inhibitor, IGF-binding protein,epidermal growth factor, growth hormone-releasing factor, annexin Vfusion protein, angiostatin, vascular endothelial growth factor-2,myeloid progenitor inhibitory factor-1, osteoprotegerin,α-1-antitrypsin, Dnase II, and α-feto proteins.

Expression of GnT-III to Boost Antibody Functionality

The addition of N-acetylglucosamine residues to the GlcNAcMan₃GlcNAc₂structure by N-acetylglucosaminyltransferases II and III yields aso-called bisected N-glycan GlcNAc₃Man₃GlcNAc₂ (FIG. 15). This structurehas been implicated in greater antibody-dependent cellular cytotoxicity(ADCC) (Umana et al. (1999) Nat. Biotechnol. 17(2):176-80).Re-engineering glycoforms of immunoglobulins expressed by mammaliancells is a tedious and cumbersome task. Especially in the case ofGnTIII, where over-expression of this enzyme has been implicated ingrowth inhibition, methods involving regulated (inducible) geneexpression had to be employed to produce immunoglobulins with bisectedN-glycans (Umana et al. (1999) Biotechnol Bioeng. 65(5):542-9; Umana etal. (1999) Nat. Biotechnol. 17(2):176-80); Umana et al. WO 03/011878;U.S. Pat. No. 6,602,684.

Accordingly, in another embodiment, the invention provides systems andmethods for producing human-like N-glycans having bisectingN-acetylglucosamine (GlcNAc) on a trimannose or pentamannose corestructure. In a preferred embodiment, the invention provides a systemand method for producing immunoglobulins having bisected N-glycans. Thesystems and methods described herein will not suffer from previousproblems, e.g., cytotoxicity associated with overexpression of GnTIII orADCC, as the host cells of the invention are engineered and selected tobe viable and preferably robust cells which produce N-glycans havingsubstantially modified human-type glycoforms such as GlcNAc₂Man₃GlcNAc₂.Thus, addition of a bisecting N-acetylglucosamine in a host cell of theinvention will have a negligible effect on the growth-phenotype orviability of those host cells.

In addition, work by others has shown that there is no linearcorrelation between GnTIII expression levels and the degree of ADCC.Umana et al. (1999) Nature Biotechnol. 17:176-80. Thus, finding theoptimal expression level in mammalian cells and maintaining itthroughout an FDA approved fermentation process seems to be a challenge.However, in cells of the invention, such as fungal cells, finding apromoter of appropriate strength to establish a robust, reliable andoptimal GnTIII expression level is a comparatively easy task for one ofskill in the art.

A host cell such as a yeast strain capable of producing glycoproteinswith bisecting N-glycans is engineered according to the invention, byintroducing into the host cell a GnTIII activity (Example 12).Preferably, the host cell is transformed with a nucleic acid thatencodes GnTIII (see, e.g., FIG. 24) or a domain thereof having enzymaticactivity, optionally fused to a heterologous cell signal targetingpeptide (e.g., using the libraries and associated methods of theinvention.) Host cells engineered to express GnTIII will produce higherantibody titers than mammalian cells are capable of. They will alsoproduce antibodies with higher potency with respect to ADCC.

Antibodies produced by mammalian cell lines transfected with GnTIII havebeen shown to be as effective as antibodies produced by non-transfectedcell-lines, but at a 10-20 fold lower concentration (Davies et al.(2001) Biotechnol. Bioeng. 74(4):288-94). An increase of productivity ofthe production vehicle of the invention over mammalian systems by afactor of twenty, and a ten-fold increase of potency will result in anet-productivity improvement of two hundred. The invention thus providesa system and method for producing high titers of an antibody having highpotency (e.g., up to several orders of magnitude more potent than whatcan currently be produced). The system and method is safe and provideshigh potency antibodies at low cost in short periods of time. Host cellsengineered to express GnTIII according to the invention produceimmunoglobulins having bisected N-glycans at rates of at least 50mg/liter/day to at least 500 mg/liter/day. In addition, eachimmunoglobulin (Ig) molecule (comprising bisecting GlcNAcs) is morepotent than the same Ig molecule produced without bisecting GlcNAcs.

The following are examples which illustrate various aspects of theinvention. These examples should not be construed as limiting: theexamples are included for the purposes of illustration only.

Example 1 Cloning and Disruption of the OCH1 Gene in P. pastoris

Generation of an OCH1 Mutant of P. pastoris:

A 1215 bp ORF of the P. pastoris OCH1 gene encoding a putative α-1,6mannosyltransferase was amplified from P. pastoris genomic DNA (strainX-33, Invitrogen, Carlsbad, Calif.) using the oligonucleotides5′-ATGGCGAAGGCAGATGGCAGT-3′ (SEQ ID NO:3) and5′-TTAGTCCTTCCAACTTCCTTC-3′ (SEQ ID NO:4) which were designed based onthe P. pastoris OCH1 sequence (Japanese Patent Application PublicationNo. 8-336387). Subsequently, 2685 bp upstream and 1175 bp downstream ofthe ORF of the OCH1 gene were amplified from a P. pastoris genomic DNAlibrary (Boehm, T. et al. (1999) Yeast 15(7):563-72) using the internaloligonucleotides 5′-ACTGCCATCTGCCTTCGCCAT-3′ (SEQ ID NO:47) in the OCH1gene, and 5′-GTAATACGACTCACTATAGGGC-3′ T7 (SEQ ID NO:48) and5′-AATTAACCCTCACTAAAGGG-3′ T3 (SEQ ID NO:49) oligonucleotides in thebackbone of the library bearing plasmid lambda ZAP II (Stratagene, LaJolla, Calif.). The resulting 5075 bp fragment was cloned into thepCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) and designated pBK9.

After assembling a gene knockout construct that substituted the OCH1reading frame with a HIS4 resistance gene, P. pastoris was transformedand colonies were screened for temperature sensitivity at 37° C. OCH1mutants of S. cerevisiae are temperature sensitive and are slow growersat elevated temperatures. One can thus identify functional homologs ofOCH1 in P. pastoris by complementing an OCH1 mutant of S. cerevisiaewith a P. pastoris DNA or cDNA library. About 20 temperature sensitivestrains were further subjected to a colony PCR screen to identifycolonies with a deleted och1 gene. Several och1 deletions were obtained.

The linearized pBK9.1, which has 2.1 kb upstream sequence and 1.5 kbdownstream sequence of OCH1 gene cassette carrying Pichia HIS4 gene, wastransformed into P. pastoris BK1 [GS115 (his4 Invitrogen Corp., SanDiego, Calif.) carrying the human IFN-β gene in the AOX1 locus] to knockout the wild-type OCH1 gene. The initial screening of transformants wasperformed using histidine drop-out medium followed by replica plating toselect the temperature sensitive colonies. Twenty out of two hundredhistidine-positive colonies showed a temperature sensitive phenotype at37° C. To exclude random integration of pBK9.1 into the Pichia genome,the 20 temperature-sensitive isolates were subjected to colony PCR usingprimers specific to the upstream sequence of the integration site and toHIS4 ORF. Two out of twenty colonies were och1 defective and furtheranalyzed using a Southern blot and a Western blot indicating thefunctional och1 disruption by the och1 knock-out construct. Genomic DNAwere digested using two separate restriction enzymes BglII and ClaI toconfirm the och1 knock-out and to confirm integration at the openreading frame. The Western Blot showed och1 mutants lacking a discreteband produced in the GS 115 wild type at 46.2 kDa.

Example 2 Engineering of P. pastoris with α-1,2-Mannosidase to ProduceMan₅GlcNAc₂-Containing IFN-β Precursors

An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂ toyield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. While the production of a Man₅GlcNAc₂ precursor is essential,it is not necessarily sufficient for the production of hybrid andcomplex glycans because the specific isomer of Man₅GlcNAc₂ may or maynot be a substrate for GnTI. An och1 mutant of P. pastoris is engineeredto express secreted human interferon-β under the control of an aoxpromoter. A DNA library is constructed by the in-frame ligation of thecatalytic domain of human mannosidase IB (an α-1,2-mannosidase) with asub-library including sequences encoding early Golgi and ER localizationpeptides. The DNA library is then transformed into the host organism,resulting in a genetically mixed population wherein individualtransformants each express interferon-β as well as a syntheticmannosidase gene from the library. Individual transformant colonies arecultured and the production of interferon is induced by addition ofmethanol. Under these conditions, over 90% of the secreted protein isglycosylated interferon-β.

Supernatants are purified to remove salts and low-molecular weightcontaminants by C₁₈ silica reversed-phase chromatography. Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-β including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon-β of the parent strain. The purified interferon-β isanalyzed by MALDI-TOF mass spectroscopy and colonies expressing thedesired form of interferon-β are identified.

Example 3 Generation of an och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnTI and GnTII for Production of a Human-LikeGlycoprotein

The 1215 bp open reading frame of the P. pastoris OCH1 gene as well as2685 bp upstream and 1175 bp downstream was amplified by PCR (see alsoWO 02/00879), cloned into the pCR2.1-TOPO vector (Invitrogen) anddesignated pBK9. To create an och1 knockout strain containing multipleauxotrophic markers, 100 μg of pJN329, a plasmid containing anoch1::URA3 mutant allele flanked with SfiI restriction sites wasdigested with SfiI and used to transform P. pastoris strain JC308(Cereghino et al. (2001) Gene 263:159-169) by electroporation. Followingincubation on defined medium lacking uracil for 10 days at roomtemperature, 1000 colonies were picked and re-streaked. URA⁺ clones thatwere unable to grow at 37° C., but grew at room temperature, weresubjected to colony PCR to test for the correct integration of theoch1::URA3 mutant allele. One clone that exhibited the expected PCRpattern was designated YJN153. The Kringle 3 domain of human plasminogen(K3) was used as a model protein. A Neo^(R) marked plasmid containingthe K3 gene was transformed into strain YJN153 and a resulting strain,expressing K3, was named BK64-1.

Plasmid pPB103, containing the Kluyveromyces lactis MNN2-2 gene whichencodes a Golgi UDP-N-acetylglucosamine transporter was constructed bycloning a blunt BglII-HindIII fragment from vector pDL02 (Abeijon et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:5963-5968) into BglII and BamHIdigested and blunt ended pBLADE-SX containing the P. pastoris ADE1 gene(Cereghino et al. (2001) Gene 263:159-169). This plasmid was linearizedwith EcoNI and transformed into strain BK64-1 by electroporation and onestrain confirmed to contain the MNN2-2 by PCR analysis was named PBP1.

A library of mannosidase constructs was generated, comprising in-framefusions of the leader domains of several type I or type II membraneproteins from S. cerevisiae and P. pastoris fused with the catalyticdomains of several α-1,2-mannosidase genes from human, mouse, fly, wormand yeast sources (see, e.g., WO02/00879, incorporated herein byreference). This library was created in a P. pastoris HIS4 integrationvector and screened by linearizing with SalI, transforming byelectroporation into strain PBP1, and analyzing the glycans releasedfrom the K3 reporter protein. One active construct chosen was a chimeraof the 988-1296 nucleotides (C-terminus) of the yeast SEC12 gene fusedwith a N-terminal deletion of the mouse α-1,2-mannosidase IA gene (FIG.3), which was missing the 187 nucleotides. A P. pastoris strainexpressing this construct was named PBP2.

A library of GnTI constructs was generated, comprising in-frame fusionsof the same leader library with the catalytic domains of GnTI genes fromhuman, worm, frog and fly sources (WO 02/00879). This library wascreated in a P. pastoris ARG4 integration vector and screened bylinearizing with AatII, transforming by electroporation into strainPBP2, and analyzing the glycans released from K3. One active constructchosen was a chimera of the first 120 bp of the S. cerevisiae MNN9 genefused to a deletion of the human GnTI gene, which was missing the first154 bp. A P. pastoris strain expressing this construct was named PBP-3.(See also FIG. 36.)

A library of GnTII constructs was generated, which comprised in-framefusions of the leader library with the catalytic domains of GnTII genesfrom human and rat sources (WO 02/00879). This library was created in aP. pastoris integration vector containing the NST^(R) gene conferringresistance to the drug nourseothricin. The library plasmids werelinearized with EcoRI, transformed into strain RDP27 by electroporation,and the resulting strains were screened by analysis of the releasedglycans from purified K3.

Materials for the Following Reactions

MOPS, sodium cacodylate, manganese chloride, UDP-galactose andCMP-N-acetylneuraminic acid were from Sigma. Trifluoroacetic acid (TFA)was from Sigma/Aldrich, Saint Louis, Mo. Recombinant ratα2,6-sialyltransferase from Spodoptera frugiperda andβ1,4-galactosyltransferase from bovine milk were from Calbiochem (SanDiego, Calif.). Protein N-glycosidase F, mannosidases, andoligosaccharides were from Glyko (San Rafael, Calif.). DEAE ToyoPearlresin was from TosoHaas. Metal chelating “HisBind” resin was fromNovagen (Madison, Wis.). 96-well lysate-clearing plates were fromPromega (Madison, Wis.). Protein-binding 96-well plates were fromMillipore (Bedford, Mass.). Salts and buffering agents were from Sigma(St. Louis, Mo.). MALDI matrices were from Aldrich (Milwaukee, Wis.).

Protein Purification

Kringle 3 was purified using a 96-well format on a Beckman BioMek 2000sample-handling robot (Beckman/Coulter Ranch Cucamonga, Calif.). Kringle3 was purified from expression media using a C-terminal hexa-histidinetag. The robotic purification is an adaptation of the protocol providedby Novagen for their HisBind resin. Briefly, a 150 uL (μL) settledvolume of resin is poured into the wells of a 96-well lysate-bindingplate, washed with 3 volumes of water and charged with 5 volumes of 50mM NiSO4 and washed with 3 volumes of binding buffer (5 mM imidazole,0.5 M NaCl, 20 mM Tris-HCL pH7.9). The protein expression media isdiluted 3:2, media/PBS (60 mM PO4, 16 mM KCl, 822 mM NaCl pH7.4) andloaded onto the columns. After draining, the columns are washed with 10volumes of binding buffer and 6 volumes of wash buffer (30 mM imidazole,0.5 M NaCl, 20 mM Tris-HCl pH7.9) and the protein is eluted with 6volumes of elution buffer (1M imidazole, 0.5 M NaCl, 20 mM Tris-HClpH7.9). The eluted glycoproteins are evaporated to dryness bylyophilyzation.

Release of N-Linked Glycans

The glycans are released and separated from the glycoproteins by amodification of a previously reported method (Papac, et al. A. J. S.(1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP(Immobilon-P membrane) plate (Millipore) are wetted with 100 uL ofmethanol, washed with 3×150 uL of water and 50 uL of RCM buffer (8Murea, 360 mM Tris, 3.2 mM EDTA pH8.6), draining with gentle vacuum aftereach addition. The dried protein samples are dissolved in 30 uL of RCMbuffer and transferred to the wells containing 10 uL of RCM buffer. Thewells are drained and washed twice with RCM buffer. The proteins arereduced by addition of 60 uL of 0.1M DTT in RCM buffer for 1 hr at 37°C. The wells are washed three times with 300 uL of water andcarboxymethylated by addition of 60 uL of 0.1M iodoacetic acid for 30min in the dark at room temperature. The wells are again washed threetimes with water and the membranes blocked by the addition of 100 uL of1% PVP 360 in water for 1 hr at room temperature. The wells are drainedand washed three times with 300 uL of water and deglycosylated by theaddition of 30 uL of 10 mM NH₄HCO₃ pH 8.3 containing one milliunit ofN-glycanase (Glyko). After 16 hours at 37° C., the solution containingthe glycans was removed by centrifugation and evaporated to dryness.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry

Molecular weights of the glycans were determined using a Voyager DE PROlinear MALDI-TOF (Applied Biosciences) mass spectrometer using delayedextraction. The dried glycans from each well were dissolved in 15 uL ofwater and 0.5 uL spotted on stainless steel sample plates and mixed with0.5 uL of S-DHB matrix (9 mg/mL of dihydroxybenzoic acid, 1 mg/mL of5-methoxysalicilic acid in 1:1 water/acetonitrile 0.1% TFA) and allowedto dry.

Ions were generated by irradiation with a pulsed nitrogen laser (337 nm)with a 4 ns pulse time. The instrument was operated in the delayedextraction mode with a 125 ns delay and an accelerating voltage of 20kV. The grid voltage was 93.00%, guide wire voltage was 0.10%, theinternal pressure was less than 5×10−7 torr, and the low mass gate was875 Da. Spectra were generated from the sum of 100-200 laser pulses andacquired with a 2 GHz digitizer. Man₅GlcNAc₂ oligosaccharide was used asan external molecular weight standard. All spectra were generated withthe instrument in the positive ion mode. The estimated mass accuracy ofthe spectra was 0.5%.

Example 4 Engineering of P. pastoris to Produce Man₅GlcNAc₂ as thePredominant N-Glycan Structure Using a Combinatorial DNA Library

An och1 mutant of P. pastoris (see Examples 1 and 3) was engineered toexpress and secrete proteins such as the kringle 3 domain of humanplasminogen (K3) under the control of the inducible AOXI promoter. TheKringle 3 domain of human plasminogen (K3) was used as a model protein.A DNA fragment encoding the K3 was amplified using Pfu turbo polymerase(Strategene, La Jolla, Calif.) and cloned into EcoRI and XbaI sites ofpPICZαA (Invitrogen, Carlsbad, Calif.), resulting in a C-terminal 6-Histag. In order to improve the N-linked glycosylation efficiency of K3(Hayes et al. 1975 J Arch. Biochem. Biophys. 171, 651-655), Pro₄₆ wasreplaced with Ser₄₆ using site-directed mutagenesis. The resultingplasmid was designated pBK64. The correct sequence of the PCR constructwas confirmed by DNA sequencing.

A combinatorial DNA library was constructed by the in-frame ligation ofmurine α-1,2-mannosidase IB (Genbank AN 6678787) and IA (Genbank AN6754619) catalytic domains with a sub-library including sequencesencoding Cop II vesicle, ER, and early Golgi localization peptidesaccording to Table 6. The combined DNA library was used to generateindividual fusion constructs, which were then transformed into the K3expressing host organism, resulting in a genetically mixed populationwherein individual transformants each express K3 as well as alocalization signal/mannosidase fusion gene from the library. Individualtransform ants were cultured and the production of K3 was induced bytransfer to a methanol containing medium. Under these conditions, after24 hours of induction, over 90% of the protein in the medium was K3. TheK3 reporter protein was purified from the supernatant to remove saltsand low-molecular weight contaminants by Ni-affinity chromatography.Following affinity purification, the protein was desalted by sizeexclusion chromatography on a Sephadex G10 resin (Sigma, St. Louis, Mo.)and either directly subjected to MALDI-TOF analysis described below orthe N-glycans were removed by PNGase digestion as described below(Release of N-glycans) and subjected to MALDI-TOF analysis Miele et al.(1997) Biotechnol. Appl. Biochem. 25:151-157.

Following this approach, a diverse set of transformants were obtained;some showed no modification of the N-glycans compared to the och1knockout strain; and others showed a high degree of mannose trimming(FIGS. 5D and 5E). Desired transformants expressing appropriatelytargeted, active α-1,2-mannosidase produced K3 with N-glycans of thestructure Man₅GlcNAc₂. This confers a reduced molecular mass to theglycoprotein compared to the K3 of the parent och1 deletion strain, adifference which was readily detected by MALDI-TOF mass spectrometry(FIG. 5). Table 7 indicates the relative Man₅GlcNAc₂ production levels.

TABLE 7 A representative combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences MNS1(s) MNS1(m) MNS1(I) SEC12(s)SEC12(m) Catalytic Domians Mouse mannosidase FB4 FB5 FB6 FB7 FB8 1A Δ187++ + − ++ ++++ Mouse mannosidase GB4 GB5 GB6 GB7 GB8 1B Δ58 ++ + + ++ +Mouse mannosidase GC4 GC5 GC6 GC7 GC8 1B Δ99 − +++ + + + Mousemannosidase GD4 GD5 GD6 GD7 GD8 1B Δ170 − − − + +

TABLE 8 Another combinatorial DNA library of localizationsequences/catalytic domains exhibiting relative levels of Man₅GlcNAc₂production. Targeting peptide sequences VAN1(s) VAN1(m) VANi(I) MNN10(s)MNN10(m) MNN10(I) Catalytic Domians C. elegans BC18-5 BC19 BC20 BC27BC28 BC29 mannosidase +++++ ++++ +++ +++++ +++++ +++ 1B Δ80 C. elegansBB18 BB19 BB20 BB18 BB19 BB20 mannosidase +++++ +++++ ++++ +++++ +++++++++ 1B Δ31

Targeting peptides were selected from MNS I (SwissProt P32906) in S.cerevisiae (long, medium and short) (see supra Nucleic Acid Libraries;Combinatorial DNA Library of Fusion Constructs) and SEC12 (SwissProtP11655) in S. cerevisiae (988-1140 nucleotides: short) and (988-1296:medium). Although majority of the targeting peptide sequences wereN-terminal deletions, some targeting peptide sequences, such as SEC12were C-terminal deletions. Catalytic domains used in this experimentwere selected from mouse mannosidase 1A with a 187 amino acid N-terminaldeletion; and mouse mannosidase 1B with a 58, 99 and 170 amino aciddeletion. The number of (+)s, as used herein, indicates the relativelevels of Man₅GlcNAc₂ production. The notation (−) indicates no apparentproduction of Man₅GlcNAc₂. The notation (+) indicates less than 10%production of Man₅GlcNAc₂ The notation (++) indicates about 10-20%production of Man₅GlcNAc₂. The notation with (+++) indicates about20-40% production of Man₅GlcNAc₂. The notation with (++++) indicatesabout 50% production of Man₅GlcNAc₂. The notation with (+++++) indicatesgreater than 50% production of Man₅GlcNAC₂.

Table 9 shows relative amount of Man₅GlcNAc₂ on secreted K3. Six hundredand eight (608) different strains of P. pastoris, Δoch1 were generatedby transforming them with a single construct of a combinatorial geneticlibrary that was generated by fusing nineteen (19) α-1,2 mannosidasecatalytic domains to thirty-two (32) fungal ER, and cis-Golgi leaders.

TABLE 9 Amount of Man₅GlcNAc₂ on Number of secreted K3 (% of totalglycans) constructs (%) N.D.*  19 (3.1)  0-10% 341 (56.1) 10-20%  50(8.2) 20-40&  75 (12.3) 40-60%  72 (11.8) More than 60%  51 (8.4)^(†)Total 608 (100) *Several fusion constructs were not tested because thecorresponding plasmids could not be propagated in E. coli prior totransformation into P. pastoris. ^(†)Clones with the highest degree ofMan₅GlcNAc₂ trimming (30/51) were further analyzed for mannosidaseactivity in the supernatant of the medium. The majority (28/30)displayed detectable mannosidase activity in the supernatant (e.g. FIG.4B). Only two constructs displayed high Man₅GlcNAc₂ levels, whilelacking mannosidase activity in the medium (e.g. FIG. 4C).

Table 7 shows two constructs pFB8 and pGC5, among others, displayingMan₅GlcNAc₂. Table 8 shows a more preferred construct, pBC18-5, a S.cerevisiae VAN1(s) targeting peptide sequence (from SwissProt 23642)ligated in-frame to a C. elegans mannosidase IB (Genbank AN CAA98114) 80amino acid N-terminal deletion (Saccharomyces Van1(s)/C. elegansmannosidase IB Δ80). This fusion construct also produces a predominantMan₅GlcNAc₂ structure, as shown in FIG. 5E. This construct was shown toproduce greater than 50% Man₅GlcNAc₂ (+++++).

Generation of a Combinatorial Localization/Mannosidase Library:

Generating a combinatorial DNA library of α-1,2-mannosidase catalyticdomains fused to targeting peptides required the amplification ofmannosidase domains with varying lengths of N-terminal deletions from anumber of organisms. To approach this goal, the full length open readingframes (ORFs) of α-1,2-mannosidases were PCR amplified from either cDNAor genomic DNA obtained from the following sources: Homo sapiens, Musmusculus, Drosophila melanogaster, Caenorhabditis elegans, Aspergillusnidulans and Penicillium citrinum. In each case, DNA was incubated inthe presence of oligonucleotide primers specific for the desiredmannosidase sequence in addition to reagents required to perform the PCRreaction. For example, to amplify the ORF of the M. musculusα-1,2-mannosidase IA, the 5′-primer ATGCCCGTGGGGGGCCTGTTGCCGCTCTTCAGTAGC(SEQ ID NO:52) and the 3′-primerTCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG (SEQ ID NO:53) were incubatedin the presence of Pfu DNA polymerase (Stratagene, La Jolla, Calif.) andamplified under the conditions recommended by Stratagene using thecycling parameters: 94° C. for 1 min (1 cycle); 94° C. for 30 sec, 68°C. for 30 sec, 72° C. for 3 min (30 cycles). Following amplification theDNA sequence encoding the ORF was incubated at 72° C. for 5 min with 1 UTaq DNA polymerase (Promega, Madison, Wis.) prior to ligation intopCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) and transformed into TOP 10chemically competent E. coli, as recommended by Invitrogen. The clonedPCR product was confirmed by ABI sequencing using primers specific forthe mannosidase ORF.

To generate the desired N-terminal truncations of each mannosidase, thecomplete ORF of each mannosidase was used as the template in asubsequent round of PCR reactions wherein the annealing position of the5′-primer was specific to the 5′-terminus of the desired truncation andthe 3′-primer remained specific for the original 3′-terminus of the ORF.To facilitate subcloning of the truncated mannosidase fragment into theyeast expression vector, pJN347 (FIG. 2C) AscI and PacI restrictionsites were engineered onto each truncation product, at the 5′- and3′-termini respectively. The number and position of the N-terminaltruncations generated for each mannosidase ORF depended on the positionof the transmembrane (TM) region in relation to the catalytic domain(CD). For instance, if the stem region located between the TM and CD wasless than 150 bp, then only one truncation for that protein wasgenerated. If, however, the stem region was longer than 150 bp theneither one or two more truncations were generated depending on thelength of the stem region.

An example of how truncations for the M. musculus mannosidase IA(Genbank AN 6678787) were generated is described herein, with a similarapproach being used for the other mannosidases. FIG. 3 illustrates theORF of the M. musculus α-1,2-mannosidase IA with the predictedtransmembrane and catalytic domains being highlighted in bold. Based onthis structure, three 5′-primers were designed (annealing positionsunderlined in FIG. 3) to generate the Δ65-, Δ105- and Δ187-N-terminaldeletions. Using the Δ65 N-terninal deletion as an example the 5′-primerused was 5′-GGCGCGCCGACTCCTCCAAGCTGCTCAGCGGGGTCCTGTTCCAC-3′ (SEQ IDNO:54) (with the AscI restriction site highlighted in bold) inconjunction with the 3′-primer5′-CCTTAATTAATCATTTCTCTTTGCCATCAATTTCCTTCTTCTGTTCACGG-3′ (SEQ ID NO:55)(with the PacI restriction site highlighted in bold). Both of theseprimers were used to amplify a 1561 bp fragment under the conditionsoutlined above for amplifying the full length M. musculus mannosidase 1AORF. Furthermore, like the product obtained for the full length ORF, thetruncated product was also incubated with Taq DNA polymerase, ligatedinto pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.), transformed into TOP10and ABI sequenced. After having amplified and confirmed the sequence ofthe truncated mannosidase fragment, the resulting plasmid, pCR2.1-Δ65mMannIA, was digested with AscI and PacI in New England Biolabs buffer#4 (Beverly, Mass.) for 16 h at 37° C. In parallel, the pJN347 (FIG. 2C)was digested with the same enzymes and incubated as described above.Post-digestion, both the pJN347 (FIG. 2C) back-bone and the truncatedcatalytic domain were gel extracted and ligated using the Quick LigationKit (New England Biolabs, Beverly, Mass.), as recommended by themanufacturers, and transformed into chemically competent DH5α cells(Invitrogen, Carlsbad, Calif.). Colony PCR was used to confirm thegeneration of the pJN347-mouse Mannosidase IAΔ65 construct.

Having generated a library of truncated α-1,2-mannosidase catalyticdomains in the yeast expression vector pJN347 (FIG. 2C) the remainingstep in generating the targeting peptide/catalytic domain library was toclone in-frame the targeting peptide sequences (FIG. 2). Both thepJN347-mannosidase constructs (FIG. 2D) and the pCR2.1TOPO-targetingpeptide constructs (FIG. 2B) such as were incubated overnight at 37° C.in New England Biolabs buffer #4 in the presence of the restrictionenzymes NotI and AscI. Following digestion, both the pJN347-mannosidaseback-bone and the targeting peptide regions were gel-extracted andligated using the Quick Ligation Kit (New England Biolabs, Beverly,Mass.), as recommended by the manufacturers, and transformed intochemically competent DH5α cells (Invitrogen, Carlsbad, Calif.).Subsequently, the pJN347-targeting peptide/mannosidase constructs wereABI sequenced to confirm that the generated fusions were in-frame. Theestimated size of the final targeting peptide/alpha-1,2-mannosidaselibrary contains over 1300 constructs generated by the approachdescribed above. FIG. 2 illustrates construction of the combinatorialDNA library.

Engineering a P. pastoris OCH1 Knock-Out Strain with MultipleAuxotrophic Markers

The first step in plasmid construction involved creating a set ofuniversal plasmids containing DNA regions of the KEX1 gene of P.pastoris (Boehm et al. Yeast 1999 May; 15(7):563-72) as space holdersfor the 5′ and 3′ regions of the genes to be knocked out. The plasmidsalso contained the S. cerevisiae Ura-blaster (Alani et al. (1987)Genetics 116:541-545) as a space holder for the auxotrophic markers, andan expression cassette with a multiple cloning site for insertion of aforeign gene. A 0.9-kb fragment of the P. pastoris KEX1-5′ region wasamplified by PCR using primers

GGCGAGCTCGGCCTACCCGGCCAAGGCTGAGATCATTTGTCCAGCTTCAGA (SEQ ID NO:56) and

GCCCACGTCGACGGATCCGTTTAAACATCGATTGGAGAGGCTGACACCGCTACTA (SEQ ID NO:57)and P. pastoris genomic DNA as a template and cloned into the SacI, SalIsites of pUC19 (New England Biolabs, Beverly, Mass.). The resultingplasmid was cut with BamHI and SalI, and a 0.8-kb fragment of theKEX1-3′ region that had been amplified using primersCGGGATCCACTAGTATTTAAATCATATGTGCGAGTGTACAACTCTTCCCACATGG (SEQ ID NO:58)andGGACGCGTCGACGGCCTACCCGGCCGTACGAGGAATTTCTCGGATGACTCTTTTC (SEQ ID NO:59)was cloned into the open sites creating pJN262. This plasmid was cutwith BamHI and the 3.8-kb BamHI, BglII fragment of pNKY51 (Alani et al.(1987) Genetics 116:541-545) was inserted in both possible orientationsresulting in plasmids pJN263 (FIG. 4A) and pJN284 (FIG. 4B).

An expression cassette was created with NotI and PacI as cloning sites.The GAPDH promoter of P. pastoris was amplified using primers

CGGGATCCCTCGAGAGATCTTTTTTGTAGAAATGTCTTGGTGCCT (SEQ ID NO:60) and

GGACATGCATGCACTAGTGCGGCCGCCACGTGATAGTTGTTCAATTGATTGAAATAGGGACAA (SEQ IDNO:61) and plasmid pGAPZ-A (Invitrogen) as template and cloned into theBamHI, SphI sites of pUC19 (New England Biolabs, Beverly, Mass.) (FIG.4B). The resulting plasmid was cut with SpeI and SphI and the CYC1transcriptional terminator region (“TT”) that had been amplified usingprimersCCTTGCTAGCTTAATTAACCGCGGCACGTCCGACGGCGGCCCACGGGTCCCA (SEQ ID NO:62) andGGACATGCATGCGGATCCCTTAAGAGCCGGCAGCTTGCAAATTAAAGCCTTCGAGCGTCCC (SEQ IDNO:63) and plasmid pPICZ-A (Invitrogen) as a template was cloned intothe open sites creating pJN261 (FIG. 4B).

A knockout plasmid for the P. pastoris OCH1 gene was created bydigesting pJN263 with SalI and SpeI and a 2.9-kb DNA fragment of theOCH1-5′ region, which had been amplified using the primers

GAACCACGTCGACGGCCATTGCGGCCAAAACCTTTTTTCCTATTCAAACACAAGGCATTGC (SEQ IDNO:64) and

CTCCAATACTAGTCGAAGATTATCTTCTACGGTGCCTGGACTC (SEQ ID NO:65) and P.pastoris genomic DNA as a template, was cloned into the open sites (FIG.4C). The resulting plasmid was cut with EcoRI and PmeI and a 1.0-kb DNAfragment of the OCH1-3′ region that had been generated using the primersTGGAAGGTTTAAACAAAGCTAGAGTAAAATAGATATAGCGAGATTAGAGAATG (SEQ ID NO:66) andAAGAATTCGGCTGGAAGGCCTTGTACCTTGATGTAGTTCCCGTTTTCATC (SEQ ID NO:67) wasinserted to generate pJN298 (FIG. 4C). To allow for the possibility tosimultaneously use the plasmid to introduce a new gene, the BamHIexpression cassette of pJN261 (FIG. 4B) was cloned into the unique BamHIsite of pJN298 (FIG. 4C) to create pJN299 (FIG. 4E).

The P. pastoris Ura3-blaster cassette was constructed using a similarstrategy as described in Lu et al. (1998) Appl. Microbiol. Biotechnol.49:141-146. A 2.0-kb PstI, SpeI fragment of P. pastoris URA3 wasinserted into the PstI, XbaI sites of pUC19 (New England Biolabs,Beverly, Mass.) to create pJN306 (FIG. 4D). Then a 0.7-kb SacI, PvuIIDNA fragment of the lacZ open reading frame was cloned into the SacI,SmaI sites to yield pJN308 (FIG. 4D). Following digestion of pJN308(FIG. 4D) with PstI, and treatment with T4 DNA polymerase, theSacI-PvuII fragment from lacZ that had been blunt-ended with T4 DNApolymerase was inserted generating pJN315 (FIG. 4D). The lacZ/URA3cassette was released by digestion with SacI and SphI, blunt ended withT4 DNA polymerase and cloned into the backbone of pJN299 that had beendigested with PmeI and AflII and blunt ended with T4 DNA polymerase. Theresulting plasmid was named pJN329 (FIG. 4E).

A HIS4 marked expression plasmid was created by cutting pJN261 (FIG. 4F)with EcoICRI (FIG. 4F). A 2.7 kb fragment of the Pichia pastoris HIS4gene that had been amplified using the primers

GCCCAAGCCGGCCTTAAGGGATCTCCTGATGACTGACTCACTGATAATAAAAATACGG (SEQ IDNO:68) and

GGGCGCGTATTTAAATACTAGTGGATCTATCGAATCTAAATGTAAGTTAAAATCTCTAA (SEQ IDNO:69) cut with NgoMIV and SwaI and then blunt-ended using T4 DNApolymerase, was then ligated into the open site. This plasmid was namedpJN337 (FIG. 4F). To construct a plasmid with a multiple cloning sitesuitable for fusion library construction, pJN337 was cut with NotI andPacI and the two oligonucleotidesGGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT (SEQ ID NO:70) andTAAGGCGCGCCGAATTCATTTAAATCTGCAGGGC (SEQ ID NO:71) that had been annealedin vitro were ligated into the open sites, creating pJN347 (FIG. 4F).

To create an och1 knockout strain containing multiple auxotrophicmarkers, 100 μg of pJN329 was digested with SfiI and used to transformP. pastoris strain JC308 (Cereghino et al. (2001) Gene 263:159-169) byelectroporation. Following transformation, the URA dropout plates wereincubated at room temperature for 10 days. One thousand (1000) colonieswere picked and restreaked. All 000 clones were then streaked onto 2sets of URA dropout plates. One set was incubated at room temperature,whereas the second set was incubated at 37° C. The clones that wereunable to grow at 37° C., but grew at room temperature, were subjectedto colony PCR to test for the correct OCH1 knockout. One clone thatshowed the expected PCR signal (about 4.5 kb) was designated YJN153.

Example 5 Characterization of the Combinatorial DNA Library

Positive transformants screened by colony PCR confirming integration ofthe mannosidase construct into the P. pastoris genome were subsequentlygrown at room temperature in 50 ml BMGY buffered methanol-complex mediumconsisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphatebuffer, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1%glycerol as a growth medium) until OD_(600nm) 2-6 at which point theywere washed with 10 ml BMMY (buffered methanol-complex medium consistingof 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin, and 1.5% methanol as agrowth medium) media prior to induction of the reporter protein for 24hours at room temperature in 5 ml BMMY. Consequently, the reporterprotein was isolated and analyzed as described in Example 3 tocharacterize its glycan structure. Using the targeting peptides in Table6, mannosidase catalytic domains localized to either the ER or the Golgishowed significant level of trimming of a glycan predominantlycontaining Man₈GlcNAc₂ to a glycan predominantly containing Man₅GlcNAc₂.This is evident when the glycan structure of the reporter glycoproteinis compared between that of P. pastoris och1 knock-out in FIGS. 5C and6C and the same strain transformed with M. musculus mannosidaseconstructs as shown in FIGS. 5D, 5E, 6D-6F. FIGS. 5 and 6 showexpression of constructs generated from the combinatorial DNA librarywhich show significant mannosidase activity in P. pastoris. Expressionof pGC5 (Saccharomyces MNS1(m)/mouse mannosidase IB Δ99) (FIGS. 5D and6E) produced a protein which has approximately 30% of all glycanstrimmed to Man₅GlcNAc₂, while expression of pFB8 (SaccharomycesSEC12(m)/mouse mannosidase IA Δ187) (FIG. 6F) produced approximately 50%Man₅GlcNAc₂ and expression of pBC18-5 (Saccharomyces VAN1(s)/C. elegansmannosidase IB Δ80) (FIG. 5E) produced 70% Man₅GlcNAc₂.

Example 6 Trimming In Vivo by α-1,2-Mannosidase

To ensure that the novel engineered strains of Example 4 in factproduced the desired Man₅GlcNAc₂ structure in vivo, cell supernatantswere tested for mannosidase activity (see FIGS. 7-9). For eachconstruct/host strain described below, HPLC was performed at 30° C. witha 4.0 mm×250 mm column of Altech (Avondale, Pa., USA) Econosil-NH₂ resin(5 μm) at a flow rate of 1.0 ml/min for 40 min. In FIGS. 7 and 8,degradation of the standard Man₉GlcNAc₂ [b] was shown to occur resultingin a peak which correlates to Man₈GlcNAc₂. In FIG. 7, the Man₉GlcNAc₂[b] standard eluted at 24.61 min and Man₅GlcNAc₂ [a] eluted at 18.59min. In FIG. 8, Man₉GlcNAc₂ eluted at 21.37 min and Man₅GlcNAc₂ at 15.67min. In FIG. 9, the standard Man₈GlcNAc₂ [b] was shown to elute at 20.88min.

P. pastoris cells comprising plasmid pFB8 (Saccharomyces SEC12 (m)/mousemannosidase IA Δ187) were grown at 30° C. in BMGY to an OD600 of about10. Cells were harvested by centrifugation and transferred to BMMY toinduce the production of K3 (kringle 3 from human plasminogen) undercontrol of an AOX1 promoter. After 24 hours of induction, cells wereremoved by centrifugation to yield an essentially clear supernatant. Analiquot of the supernatant was removed for mannosidase assays and theremainder was used for the recovery of secreted soluble K3. A singlepurification step using CM-sepharose chromatography and an elutiongradient of 25 mM NaAc, pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted ina 95% pure K3 eluting between 300-500 mM NaCl. N-glycan analysis of theK3 derived glycans is shown in FIG. 6F. The earlier removed aliquot ofthe supernatant was further tested for the presence of secretedmannosidase activity. A commercially available standard of2-aminobenzamide-labeled N-linked-type oligomannose 9 (Man9-2-AB)(Glyko, Novato, Calif.) was added to: BMMY (FIG. 7A), the supernatantfrom the above aliquot (FIG. 7B), and BMMY containing 10 ng of 75 mU/mLof α-1,2-mannosidase from Trichoderma reesei (obtained from Contreras etal., WO 02/00856 A2) (FIG. 7C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming.

P. pastoris cells comprising plasmid pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) were similarly grown and assayed. Cells were grownat room temperature in BMGY to an OD600 of about 10. Cells wereharvested by centrifugation and transferred to BMMY to induce theproduction of K3 under control of an AOX1 promoter. After 24 hours ofinduction, cells were removed by centrifugation to yield an essentiallyclear supernatant. An aliquot of the supernatant was removed formannosidase assays and the remainder was used for the recovery ofsecreted soluble K3. A single purification step using CM-sepharosechromatography and an elution gradient of 25 mM NaAc, pH5.0 to 25 mMNaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 eluting between 300-500mM NaCl. N-glycan analysis of the K3 derived glycans is shown in FIG.5D. The earlier removed aliquot of the supernatant was further testedfor the presence of secreted mannosidase activity as shown in FIG. 8B. Acommercially available standard of Man9-2-AB (Glyko, Novato, Calif.)were added to: BMMY (FIG. 8A), supernatant from the above aliquot (FIG.8B), and BMMY containing 10 ng of 75 mU/mL of α-1,2-mannosidase fromTrichoderma reesei (obtained from Contreras et al., WO 02/00856 A2)(FIG. 8C). After incubation for 24 hours at room temperature, sampleswere analyzed by amino silica HPLC to determine the extent ofmannosidase trimming.

Man9-2-AB was used as a substrate and it is evident that after 24 hoursof incubation, mannosidase activity was virtually absent in thesupernatant of the pFB8 (Saccharomyces SEC12(m)/mouse mannosidase IAΔ187) strain digest (FIG. 7B) and pGC5 (Saccharomyces MNS1(m)/mousemannosidase IB Δ99) strain digest (FIG. 8B) whereas the positive control(purified α-1,2-mannosidase from T. reesei obtained from Contreras)leads to complete conversion of Man₉GlcNAc₂ to Man₅GlcNAc₂ under thesame conditions, as shown in FIGS. 7C and 8C. This is conclusive datashowing in vivo mannosidase trimming in P. pastoris pGC5 strain; andpFB8 strain, which is distinctly different from what has been reportedto date (Contreras et al., WO 02/00856 A2) .

FIG. 9 further substantiates localization and activity of themannosidase enzyme. P. pastoris comprising pBC18-5 (SaccharomycesVAN1(s)/C. elegans mannosidase IB Δ80) was grown at room temperature inBMGY to an OD600 of about 10. Cells were harvested by centrifugation andtransferred to BMMY to induce the production of K3 under control of anAOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant. An aliquot ofthe supernatant was removed for mannosidase assays and the remainder wasused for the recovery of secreted soluble K3. A single purification stepusing CM-sepharose chromatography and an elution gradient 25 mM NaAc,pH5.0 to 25 mM NaAc, pH5.0, 1M NaCl, resulted in a 95% pure K3 elutingbetween 300-500 mM NaCl. N-glycan analysis of the K3 derived glycans isshown in FIG. 5E. The earlier removed aliquot of the supernatant wasfurther tested for the presence of secreted mannosidase activity asshown in FIG. 9B. A commercially available standard of Man8-2-AB (Glyko,Novato, Calif.) was added to: BMMY (FIG. 9A), supernatant from the abovealiquot pBC18-5 (Saccharomyces VAN1(s)/C. elegans mannosidase IB Δ80)(FIG. 9B), and BMMY containing media from a different fusion constructpDD28-3 (Saccharomyces MNN10(m) (from SwissProt 50108)/H. sapiensmannosidase IB Δ99) (FIG. 9C). After incubation for 24 hours at roomtemperature, samples were analyzed by amino silica HPLC to determine theextent of mannosidase trimming. FIG. 9B demonstrates intracellularmannosidase activity in comparison to a fusion construct pDD28-3(Saccharomyces MNN10(m) H. sapiens mannosidase IB Δ99) exhibiting anegative result (FIG. 9C).

Example 7 pH Optimum Assay of Engineered α-1,2-Mannosidase

P. pastoris cells comprising plasmid pBB27-2 (Saccharomyces MNN10(s)(from SwissProt 50108)/C. elegans mannosidase IB Δ31) were grown at roomtemperature in BMGY to an OD600 of about 17. About 80 μL of these cellswere inoculated into 600 μL BMGY and were grown overnight. Subsequently,cells were harvested by centrifugation and transferred to BMMY to inducethe production of K3 (kringle 3 from human plasminogen) under control ofan AOX1 promoter. After 24 hours of induction, cells were removed bycentrifugation to yield an essentially clear supernatant (pH 6.43). Thesupernatant was removed for mannosidase pH optimum assays.Fluorescence-labeled Man₈GlcNAc₂ (0.5 μg) was added to 20 μL ofsupernatant adjusted to various pH (FIG. 11) and incubated for 8 hoursat room temperature. Following incubation the sample was analyzed byHPLC using an Econosil NH2 4.6×250 mm, 5 micron bead, amino-bound silicacolumn (Altech, Avondale, Pa.). The flow rate was 1.0 ml/min for 40 minand the column was maintained to 30° C. After eluting isocratically (68%A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60%B) was employed over 27 min to elute the glycans (18). Solvent A(acetonitrile) and solvent B (ammonium formate, 50 mM, pH 4.5. Thecolumn was equilibrated with solvent (68% A:32% B) for 20 min betweenruns.

Example 8 Engineering of P. pastoris to Produce N-Glycans with theStructure GlcNAcMan₅GlcNAc₂

GlcNAc Transferase I activity is required for the maturation of complexand hybrid N-glycans (U.S. Pat. No. 5,834,251). Man₅GlcNAc₂ may only betrimmed by mannosidase II, a necessary step in the formation of humanglycoforms, after the addition of N-acetylglucosamine to the terminalα-1,3 mannose residue of the trimannose stem by GlcNAc Transferase I(Schachter, 1991 Glycobiology 1(5):453-461). Accordingly, acombinatorial DNA library was prepared including DNA fragments encodingsuitably targeted catalytic domains of GlcNAc Transferase I genes fromC. elegans and Homo sapiens; and localization sequences from GLS, MNS,SEC, MNN9, VAN1, ANP1, HOC1, MNN10, MNN11, MNT1, KTR1, KTR2, MNN2, MNN5,YUR1, MNN1, and MNN6 from S. cerevisiae and P. pastoris putativeα-1,2-mannosyltransferases based on the homology from S. cerevisiae: D2,D9 and J3, which are KTR homologs. Table 10 includes but does not limittargeting peptide sequences such as SEC and OCH1, from P. pastoris andK. lactis GnTI, (See Table 6 and Table 10)

TABLE 10 A representative combinatorial library of targeting peptidesequences/ catalytic domain for UDP-N-Acetylglucosaminyl Transferase I(GnTI) Targeting peptide OCHI(s) OCHI(m) OCHI(l) MNN9(s) MNN9(m)Catalytic Human, GnTI, Δ38 PB105 PB106 PB107 PB104 N/A Domain Human,GnTI, Δ86 NB12 NB13 NB14 NB15 NB C. elegans, GnTI, Δ88 OA12 OA13 OA14OA15 OA16 C. elegans, GnTI, Δ35 PA12 PA13 PA14 PA15 PA16 C. elegans,GnTI, Δ63 PB12 PB13 PB14 PB15 PB16 X. leavis, GnTI, Δ33 QA12 QA13 QA14QA15 QA16 X. leavis, GnTI, Δ103 QB12 QB13 QB14 QB15 QB16

Targeting peptide sequences were selected from OCH1 in P. pastoris(long, medium and short) (see Example 4) and MNN9 (SwissProt P39107) inS. cerevisiae (short and medium). Catalytic domains were selected fromhuman GnTI with a 38 and 86 amino acid N-terminal deletion, C. elegans(gly-12) GnTI with a 35 and 63 amino acid deletion as well as C. elegans(gly-14) GnTI with a 88 amino acid N-terminal deletion and X. leavisGnTI with a 33 and 103 amino acid N-terminal deletion, respectively.

A portion of the gene encoding human N-acetylglucosaminyl Transferase I(MGATI, Accession# NM002406), lacking the first 154 bp, was amplified byPCR using oligonucleotides 5′-TGGCAGGCGCGCCTCAGTCAGCGCTCTCG-3′ (SEQ IDNO:72) and 5′-AGGTTAATTA AGTGCTAATTCCAGCTAGG-3′ (SEQ ID NO:73) andvector pHG4.5 (ATCC# 79003) as template. The resulting PCR product wascloned into pCR2.1-TOPO and the correct sequence was confirmed.Following digestion with AscI and PacI the truncated GnTI was insertedinto plasmid pJN346 to create pNA. After digestion of pJN271 with NotIand AscI, the 120 bp insert was ligated into pNA to generate an in-framefusion of the MNN9 transmembrane domain with the GnTI, creating pNA 15.

The host organism is a strain of P. pastoris that is deficient inhypermannosylation (e.g. an och1 mutant), provides the substrateUDP-GlcNAc in the Golgi and/or ER (i.e., contains a functionalUDP-GlcNAc transporter), and provides N-glycans of the structureMan₅GlcNAc₂ in the Golgi and/or ER (e.g. P. pastoris pFB8 (SaccharomycesSEC12(m)/mouse mannosidase IA Δ187) from above). First, P. pastoris pFB8was transformed with pPB103 containing the Kluyveromyces lactis MNN2-2gene (Genbank AN AF106080) (encoding UDP-GlcNAc transporter) cloned intoBamHI and BglII site of pBLADE-SX plasmid (Cereghino et al. (2001) Gene263:159-169). Then the aforementioned combinatorial DNA library encodinga combination of exogenous or endogenous GnTI/localization genes wastransformed and colonies were selected and analyzed for the presence ofthe GnTI construct by colony PCR. Our transformation and integrationefficiency was generally above 80% and PCR screening can be omitted oncerobust transformation parameters have been established.

Protein Purification

K3 was purified from the medium by Ni-affinity chromatography utilizinga 96-well format on a Beckman BioMek 2000 laboratory robot. The roboticpurification is an adaptation of the protocol provided by Novagen fortheir HisBind resin. Another screening method may be performed using aspecific terminal GlcNAc binding antibody, or a lectin such as the GSIIlectin from Griffonia simplificolia, which binds terminal GlcNAc (EYLaboratories, San Mateo, Calif.). These screens can be automated byusing lectins or antibodies that have been modified with fluorescentlabels such as FITC or analyzed by MALDI-TOF.

Secreted K3 can be purified by Ni-affinity chromatography, quantifiedand equal amounts of protein can be bound to a high protein binding96-well plate. After blocking with BSA, plates can be probed with aGSII-FACS lectin and screened for maximum fluorescent response. Apreferred method of detecting the above glycosylated proteins involvesthe screening by MALDI-TOF mass spectrometry following the affinitypurification of secreted K3 from the supernatant of 96-well culturedtransformants. Transformed colonies were picked and grown to an OD600 of10 in a 2 ml, 96-well plate in BMGY at 30° C. Cells were harvested bycentrifugation, washed in BMMY and resuspended in 250 ul of BMMY.Following 24 hours of induction, cells were removed by centrifugation,the supernatant was recovered and K3 was purified from the supernatantby Ni affinity chromatography. The N-glycans were released and analyzedby MALDI-TOF delayed extraction mass spectrometry as described herein.

In summary, the methods of the invention yield strains of P. pastoristhat produce GlcNAcMan₅GlcNAc₂ in high yield, as shown in FIG. 10B. Atleast 60% of the N-glycans are GlcNAcMan₅GlcNAc₂. To date, no reportexists that describes the formation of GlcNAcMan₅GlcNAc₂ on secretedsoluble glycoproteins in any yeast. Results presented herein show thataddition of the UDP-GlcNAc transporter along with GnTI activity producesa predominant GlcNAcMan₅GlcNAc₂ structure, which is confirmed by thepeak at 1457 (m/z) (FIG. 10B).

Construction of Strain PBP-3:

The P. pastoris strain expressing K3, (Δoch1, arg-, ade-, his-) wastransformed successively with the following vectors. First, pFB8(Saccharomyces SEC12(m)/mouse mannosidase IA Δ187) was transformed inthe P. pastoris strain by electroporation. Second, pPB103 containingKluyveromyces lactis MNN2-2 gene (Genbank AN AF106080) (encodingUDP-GlcNAc transporter) cloned into pBLADE-SX plasmid (Cereghino et al.(2001) Gene 263:159-169) digested with BamHI and BglII enzymes wastransformed in the P. pastoris strain. Third, pPB104 containingSaccharomyces MNN9(s)/human GnTI Δ38 encoding gene cloned as NotI-PacIfragment into pJN336 was transformed into the P. pastoris strain.

Example 9 Engineering K. lactis Cells to Produce N-Glycans with theStructure Man₅GlcNAC₂

Identification and Disruption of the K. lactis OCH1 Gene

The OCH1 gene of the budding yeast S. cerevisiae encodes a1,6-mannosyltransferase that is responsible for the first Golgilocalized mannose addition to the Man₈GlcNAc₂ N-glycan structure onsecreted proteins (Nakanishi-Shindo et al. (1993) J. Biol. Chem.;268(35):26338-45). This mannose transfer is generally recognized as thekey initial step in the fungal specific polymannosylation of N-glycanstructures (Nakanishi-Shindo et al. (1993) J. Biol. Chem.268(35):26338-26345; Nakayama et al. (1992) EMBO J. 11(7):2511-19;Morin-Ganet et al (2000) Traffic 1(1):56-68). Deletion of this gene inS. cerevisiae results in a significantly shorter N-glycan structure thatdoes not include this typical polymannosylation or a growth defect atelevated temperatures (Nakayama et al. (1992) EMBO J. 11 (7):2511-19).

The Och1p sequence from S. cerevisiae was aligned with known homologsfrom Candida albicans (Genbank accession # AAL49987), and P. pastorisalong with the Hoc1 proteins of S. cerevisiae (Neiman et al (1997)Genetics 145(3):637-45 and K. lactis (PENDANT EST database) which arerelated but distinct mannosyltransferases. Regions of high homology thatwere in common among Och1p homologs but distinct from the Hoc1p homologswere used to design pairs of degenerate primers that were directedagainst genomic DNA from the K. lactis strain MG1/2 (Bianchi et al(1987) Current Genetics 12:185-192). PCR amplification with primersRCD33 (CCAGAAGAATTCAATTYTGYCARTGG) (SEQ ID NO:74) and RCD34(CAGTGAAAATACCTGGNCCNGTCCA) (SEQ ID NO:75) resulted in a 302 bp productthat was cloned and sequenced and the predicted translation was shown tohave a high degree of homology to Och1 proteins (>55% to S. cerevisiaeOch1p).

The 302 bp PCR product was used to probe a Southern blot of genomic DNAfrom K. lactis strain (MG1/2) with high stringency (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Hybridization wasobserved in a pattern consistent with a single gene indicating that this302 bp segment corresponds to a portion of the K. lactis genome and K.lactis (KlOCH1) contains a single copy of the gene. To clone the entireKlOCH1 gene, the Southern blot was used to map the genomic locus.Accordingly, a 5.2 kb BamHI/PstI fragment was cloned by digestinggenomic DNA and ligating those fragments in the range of 5.2 kb into pUC19 (New England Biolabs, Beverly, Mass.) to create a K. lactissubgenomic library. This subgenomic library was transformed into E. coliand several hundred clones were tested by colony PCR using RCD 33/34.The 5.2 kb clone containing the predicted KlOCH1 gene was sequenced andan open reading frame of 1362 bp encoding a predicted protein that is46.5% identical to the S. cerevisiae OCH1 gene. The 5.2 kb sequence wasused to make primers for construction of an och1::KAN^(R) deletionallele using a PCR overlap method (Davidson et al. (2002) Microbiol.148(Pt 8):2607-15). This deletion allele was transformed into two K.lactis strains and G418 resistant colonies selected. These colonies werescreened by both PCR and for temperature sensitivity to obtain a straindeleted for the OCH1 ORF. The results of the experiment show strainswhich reveal a mutant PCR pattern, which were characterized by analysisof growth at various temperatures and N-glycan carbohydrate analysis ofsecreted and cell wall proteins following PNGase digestion. The och1mutation conferred a temperature sensitivity which allowed strains togrow at 30° C. but not at 35° C. FIG. 12A shows a MALDI-TOF analysis ofa wild type K. lactis strain producing N-glycans of Man₈GlcNAc₂ [c] andhigher.

Identification, Cloning, and Disruption of the K. lactis MNN1 Gene

S. cerevisiae MNN1 is the structural gene for the Golgiα-1,3-mannosyltransferase. The product of MNN1 is a 762-amino acid typeII membrane protein (Yip et al. (1994) Proc Natl Acad Sci USA.91(7):2723-7). Both N-linked and O-linked oligosaccharides isolated frommnn1 mutants lack α-1,3-mannose linkages (Raschke et al. (1973) J. BiolChem. 248(13):4660-66).

The Mnn1p sequence from S. cerevisiae was used to search the K. lactistranslated genomic sequences (PEDANT). One 405 bp DNA sequence encodinga putative protein fragment of significant similarity to Mnn1p wasidentified. An internal segment of this sequence was subsequently PCRamplified with primers KMN1 (TGCCATCTTTTAGGTCCAGGCCCGTTC) (SEQ ID NO:76)and KMN2 (GATCCCACGACGCATCGTATTTCTTTC), (SEQ ID NO:77) and used to probea Southern blot of genomic DNA from K. lactis strain (MG 1/2). Based onthe Southern hybridization data a 4.2 Kb BamHI-PstI fragment was clonedby generating a size-selected library as described herein. A singleclone containing the K. lactis MNN1 gene was identified by whole colonyPCR using primers KMN1 and KMN2 and sequenced. Within this clone a 2241bp ORF was identified encoding a predicted protein that was 34%identical to the S. cerevisiae MNN1 gene. Primers were designed forconstruction of a mnn1::NAT^(R) deletion allele using the PCR overlapmethod (Davidson et al. (2002) Microbiol. 148(Pt 8):2607-15).

This disruption allele was transformed into a strain of K. lactis byelectroporation and nourseothricin resistant transformants were selectedand PCR amplified for homologous insertion of the disruption allele.Strains that reveal a mutant PCR pattern may be subjected to N-glycancarbohydrate analysis of a known reporter gene.

FIG. 12B depicts the N-glycans from the K. lactis och1 mnn1 deletionstrain observed following PNGase digestion the MALDI-TOF as describedherein. The predominant peak at 1908 (m/z) indicated as [d] isconsistent with the mass of Man₉GlcNAc₂.

Additional methods and reagents which can be used in the methods formodifying the glycosylation are described in the literature, such asU.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No.6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat.No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S.Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854,U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.5,047,335. Appropriate yeast expression systems can be obtained fromsources such as the American Type Culture Collection, Rockville, Md.Vectors are commercially available from a variety of sources.

Example 10 Identification, Cloning and Deletion of the ALG3 Gene in P.pastoris and K. lactis

Degenerate primers were generated based on an alignment of Alg3 proteinsequences from S. cerevisiae, H. sapiens, and D. melanogaster and wereused to amplify an 83 bp product from P. pastoris genomic DNA:

(SEQ ID NO:78) 5′-GGTGTTTTGTTTTCTAGATCTTTGCAYTAYCARTT-3′ and (SEQ IDNO:79) 5′-AGAATTTGGTGGGTAAGAATTCCARCACCAYTCRTG-3′.The resulting PCR product was cloned into the pCR2.1 vector (Invitrogen,Carlsbad, Calif.) and sequence analysis revealed homology to knownALG3/RHK1/NOT56 homologs (Genbank NC_(—)001134.2, AF309689,NC_(—)003424.1). Subsequently, 1929 bp upstream and 2738 bp downstreamof the initial PCR product were amplified from a P. pastoris genomic DNAlibrary (Boehm (1999) Yeast 15(7):563-72) using the internaloligonucleotides 5′-CCTAAGCTGGTATGCGTTCTCTTTGCCATATC-3′ (SEQ ID NO:80)and 5′-GCGGCATAAACAATAATAGATGCTATAAAG-3′ (SEQ ID NO:81) along with T3(5′-AATTAACCCTCACTAAAGGG-3′) (SEQ ID NO:49) and T7 (5′-GTAATACGACTCACTATAGGGC-3′) (SEQ ID NO:48) (Integrated DNA Technologies,Coralville, Iowa) in the backbone of the library bearing plasmid lambdaZAP II (Stratagene, La Jolla, Calif.). The resulting fragments werecloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. From thissequence, a 1395 bp ORF was identified that encodes a protein with 35%identity and 53% similarity to the S. cerevisiae ALG3 gene (using BLASTprograms). The gene was named PpALG3.

The sequence of PpALG3 was used to create a set of primers to generate adeletion construct of the PpALG3 gene by PCR overlap (Davidson et al(2002) Microbiol. 148(Pt 8):2607-15). Primers below were used to amplify1 kb regions 5′ and 3′ of the PpALG3 ORF and the KAN^(R) gene,respectively:

RCD142 (5′-CCACATCATCCGTGCTACATATAG-3′), (SEQ ID NO:82) RCD144(5′-ACGAGGCAAGCTAAACAGATCTCGAAGTATCGAGGGTTATCCAG-3′), (SEQ ID NO:83)RCD145 (5′-CCATCCAGTGTCGAAAACGAGCCAATGGTTCATGTCTATAAATC-3′), (SEQ IDNO:84) RCD147 (5′-AGCCTCAGCGCCAACAAGCGATGG-3′), (SEQ ID NO:85) RCD143(5′-CTGGATAACCCTCGATACTTCGAGATCTGTTTAGCTTGCCTCGT-3′), (SEQ ID NO:86) andRCD146 (5′-GATTTATAGACATGAACCATTGGCTCGTTTTCGACACTGGATGG-3′). (SEQ IDNO:87)Subsequently, primers RCD142 and RCD147 were used to overlap the threeresulting PCR products into a single 3.6 kb alg3::KAN^(R) deletionallele.Identification, Cloning and Deletion of the ALG3 Gene in K. lactis.

The ALG3p sequences from S. cerevisiae, Drosophila melanogaster, Homosapiens etc were aligned with K. lactis sequences (PENDANT ESTdatabase). Regions of high homology that were in common homologs butdistinct in exact sequence from the homologs were used to create pairsof degenerate primers that were directed against genomic DNA from the K.lactis strain MG1/2 (Bianchi et al, 1987). In the case of ALG3, PCRamplification with primers KAL-1 (5′-ATCCTTTACCGATGCTGTAT-3′) (SEQ IDNO:88) and KAL-2 (5′-ATAACAGTATGTGTTACACGCGTGTAG-3′) (SEQ ID NO:89)resulted in a product that was cloned and sequenced and the predictedtranslation was shown to have a high degree of homology to Alg3pproteins (>50% to S. cerevisiae Alg3p).

The PCR product was used to probe a Southern blot of genomic DNA from K.lactis strain (MG1/2) with high stringency (Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989). Hybridization was observed in apattern consistent with a single gene. This Southern blot was used tomap the genomic loci. Genomic fragments were cloned by digesting genomicDNA and ligating those fragments in the appropriate size-range intopUC19 to create a K. lactis subgenomic library. This subgenomic librarywas transformed into E. coli and several hundred clones were tested bycolony PCR, using primers KAL-1 and KAL-2. The clones containing thepredicted KlALG3 and KlALG61 genes were sequenced and open readingframes identified.

Primers for construction of an alg3::NAT^(R) deletion allele, using aPCR overlap method (Davidson et al. (2002) Microbiol. 148(Pt8):2607-15), were designed and the resulting deletion allele wastransformed into two K. lactis strains and NAT-resistant coloniesselected. These colonies were screened by PCR and transformants wereobtained in which the ALG3 ORF was replaced with the och1::NAT^(R)mutant allele.

Example 11 Generation of an alg3 och1 Mutant Strain Expressing anα-1,2-Mannosidase, GnT1 and GnTII for Production of a Human-LikeGlycoprotein

A P. pastoris alg3::KAN^(R) deletion construct was generated asdescribed in Example 10. Approximately 5 μg of the resulting PCR productwas transformed into strain PBP-3 (see Example 3), and colonies wereselected on YPD medium containing 200 μg/ml G418. One strain out of 20screened by PCR was confirmed to contain the correct integration of thealg3::KAN^(R) mutant allele and lack the wild-type allele. This strainwas named RDP27 (FIG. 36).

A library of GnTII constructs was then generated, which was comprised ofin-frame fusions of the leader library with the catalytic domains ofGnTII genes from human and rat sources (WO 02/00879). This library wascreated in a P. pastoris integration vector containing the NST^(R) geneconferring resistance to the drug nourseothricin. The library plasmidswere linearized with EcoRI, transformed into strain RDP27 byelectroporation, and the resulting strains were screened by analysis ofthe released glycans from purified K3. A P. pastoris strain expressingthe rat GnTII fused in-frame to the S. cerevisiae MNN9(s) construct wasnamed PBP6-5 (FIG. 36).

Generation of GnTII Expression Constructs

The construction of a GnTI expression vector (pNA15) containing a humanGnTI gene fused with the N-terminal part of S. cerevisiae MNN9 gene isdescribed in Choi et al. (2003) Proc Natl Acad Sci U S A.100(9):5022-27. In a similar fashion, the rat GnTII gene was cloned. Therat GnTII gene (GenBank accession number U21662) was PCR amplified usingTakara EX Taq™ polymerase (Panvera) from rat liver cDNA library(Clontech) with RAT1 (5′-TTCCTCACTGCAGTCTTCTATAACT-3′) (SEQ ID NO:90)and RAT2 (5′-TGGAGACCATGAGGTTCCGCATCTAC-3′) (SEQ ID NO:91) primers. ThePCR product was then cloned into pCR2.1-TOPO vector (Invitrogen) andsequenced. Using this vector as a template, the AscI-PacI fragment ofGnTII, encoding amino-acids 88-443, was amplified with Pfu Turbopolymerase (Stratagene) and primers,

RAT44 (5′-TTGGCGCGCCTCCCTAGTGTACCAGTTGAACTTTG-3′) (SEQ ID NO:92) and

RAT11 (5′-GATTAATTAACTCACTGCAGTCTTCTATAACT-3′) (SEQ ID NO:93)respectively, introduced AscI and PacI restriction sites areunderlined). Following confirmation by sequencing, the catalytic domainof rat GnTII was than cloned downstream of the PMA1 promoter as aAscI-PacI fragment in pBP124. In the final step, the gene fragmentencoding the S. cerevisiae Mnn2 localization signal was cloned frompJN281 as a NotI-AscI fragment to generate an in-frame fusion with thecatalytic domain of GnTII, to generate plasmid pTC53.

Example 12 Cloning and Expression of GnTIII to Produce Bisecting GlcNAcswhich Boost Antibody Functionality

The addition of an N-acetylglucosamine to the GlcNAc₂Man₃GlcNAc₂structure by N-acetylglucosaminyltransferases III yields a so-calledbisected N-glycan (see FIG. 15). This structure has been implicated ingreater antibody-dependent cellular cytotoxicity (ADCC) (Umana et al.(1999) Nat. Biotechnol. 17(2):176-80).

A host cell such as a yeast strain capable of producing glycoproteinswith bisected N-glycans is engineered according to the invention, byintroducing into the host cell a GnTIII activity. Preferably, the hostcell is transformed with a nucleic acid that encodes GnTIII (e.g., amammalian such as the murine GnTIII shown in FIG. 24) or a domainthereof having enzymatic activity, optionally fused to a heterologouscell signal targeting peptide (e.g., using the libraries and associatedmethods of the invention.)

IgGs consist of two heavy-chains (V_(H), C_(H)1, C_(H)2 and C_(H)3 inFIG. 22), interconnected in the hinge region through three disulfidebridges, and two light chains (V_(L), C_(L) in FIG. 22). The lightchains (domains V_(L) and C_(L)) are linked by another disulfide bridgeto the CHI portion of the heavy chain and together with the C_(H)1 andV_(H) fragment make up the so-called Fab region. Antigens bind to theterminal portion of the Fab region. The Fc region of IgGs consists ofthe C_(H)3, the C_(H)2 and the hinge region and is responsible for theexertion of so-called effector functions (see below).

The primary function of antibodies is binding to an antigen. However,unless binding to the antigen directly inactivates the antigen (such asin the case of bacterial toxins), mere binding is meaningless unlessso-called effector-functions are triggered. Antibodies of the IgGsubclass exert two major effector-functions: the activation of thecomplement system and induction of phagocytosis. The complement systemconsists of a complex group of serum proteins involved in controllinginflammatory events, in the activation of phagocytes and in the lyticaldestruction of cell membranes. Complement activation starts with bindingof the C1 complex to the Fc portion of two IgGs in close proximity. C1consists of one molecule, C1q, and two molecules, C1r and C1s.Phagocytosis is initiated through an interaction between the IgG's Fcfragment and Fc-gamma-receptors (FcγRI, II and III in FIG. 22). Fcreceptors are primarily expressed on the surface of effector cells ofthe immune system, in particular macrophages, monocytes, myeloid cellsand dendritic cells.

The C_(H)2 portion harbors a conserved N-glycosylation site atasparagine 297 (Asn297). The Asn297 N-glycans are highly heterogeneousand are known to affect Fc receptor binding and complement activation.Only a minority (i.e., about 15-20%) of IgGs bears a disialylated, and3-10% have a monosialylated N-glycan (reviewed in Jefferis (2001)Biopharm. 14:19-26). Interestingly, the minimal N-glycan structure shownto be necessary for fully functional antibodies capable of complementactivation and Fc receptor binding is a pentasacharide with terminalN-acetylglucosamine residues (GlcNAc₂Man₃) (reviewed in Jefferis, R.,Glycosylation of human IgG Antibodies. BioPharm, 2001). Antibodies withless than a GlcNAc₂Man₃ N-glycan or no N-glycosylation at Asn297 mightstill be able to bind an antigen but most likely will not activate thecrucial downstream events such as phagocytosis and complementactivation. In addition, antibodies with fungal-type N-glycans attachedto Asn297 will in all likelihood solicit an immune-response in amammalian organism which will render that antibody useless as atherapeutic glycoprotein.

Cloning and Expression of GnTIII

The DNA fragment encoding part of the mouse GnTIII protein lacking theTM domain is PCR amplified from murine (or other mammalian) genomic DNAusing

forward (5′-TCCTGGCGCGCCTTCCCGAGAGAACTGGCCTCCCTC-3′) (SEQ ID NO:94) and

reversed (5′-AATTAATTAACCCTAGCCCTCCGCTGTATCCAACTTG-3′) (SEQ ID NO:95)primers. Those primers include AscI and PacI restriction sites that maybe used for cloning into the vector suitable for the fusion with leaderlibrary.

The nucleic acid (SEQ ID NO:45) and amino acid (SEQ ID NO:46) sequencesof murine GnTIII are shown in FIG. 24.

Cloning of Immunoglobulin-Encoding Sequences

Protocols for the cloning of the variable regions of antibodies,including primer sequences, have been published previously. Sources ofantibodies and encoding genes can be, among others, in vitro immunizedhuman B cells (see, e.g., Borreback et al. (1988) Proc. Natl. Acad. Sci.USA 85:3995-3999), peripheral blood lymphocytes or single human B cells(see, e.g., Lagerkvist et al. (1995) Biotechniques 18:862-869; andTerness et al. (1997) Hum. Immunol. 56:17-27) and transgenic micecontaining human immunoglobulin loci, allowing the creation of hybridomacell-lines.

Using standard recombinant DNA techniques, antibody-encoding nucleicacid sequences can be cloned. Sources for the genetic informationencoding immunoglobulins of interest are typically total RNApreparations from cells of interest, such as blood lymphocytes orhybridoma cell lines. For example, by employing a PCR based protocolwith specific primers, variable regions can be cloned via reversetranscription initiated from a sequence-specific primer hybridizing tothe IgG C_(H)1 domain site and a second primer encoding amino acids111-118 of the murine kappa constant region. The V_(H) and V_(K)encoding cDNAs can then be amplified as previously published (see, e.g.,Graziano et al. (1995) J Immunol. 155(10):4996-5002; Welschof et al.(1995) J. Immunol. Methods 179:203-214; and Orlandi et al. (1988) Proc.Natl. Acad. Sci. USA 86:3833). Cloning procedures for wholeimmunoglobulins (heavy and light chains) have also been published (see,e.g., Buckel et al. (1987) Gene 51:13-19; Recinos et al. (1994) Gene149: 385-386; Recinos et al. (1995) Gene 158:311-12). Additionalprotocols for the cloning and generation of antibody fragment andantibody expression constructs have been described in AntibodyEngineering, Kontermann and Dübel (2001), Eds., Springer Verlag: BerlinHeidelberg New York.

Fungal expression plasmids encoding heavy and light chain ofimmunoglobulins have been described (see, e.g., Abdel-Salam et al.(2001) Appl. Microbiol. Biotechnol. 56:157-164; and Ogunjimi et al.(1999) Biotechnology Letters 21:561-567). One can thus generateexpression plasmids harboring the constant regions of immunoglobulins.To facilitate the cloning of variable regions into these expressionvectors, suitable restriction sites can be placed in close proximity tothe termini of the variable regions. The constant regions can beconstructed in such a way that the variable regions can be easilyin-frame fused to them by a simple restriction-digest/ligationexperiment. FIG. 23 shows a schematic overview of such an expressionconstruct, designed in a very modular way, allowing easy exchange ofpromoters, transcriptional terminators, integration targeting domainsand even selection markers.

As shown in FIG. 23, V_(L) as well as V_(H) domains of choice can beeasily cloned in-frame with C_(L) and the C_(H) regions, respectively.Initial integration is targeted to the P. pastoris AOX locus (orhomologous locus in another fungal cell) and the methanol-inducible AOXpromoter will drive expression. Alternatively, any other desiredconstitutive or inducible promoter cassette may be used. Thus, ifdesired, the 5′ AOX and 3′ AOX regions as well as transcriptionalterminator (TT) fragments can be easily replaced with different TT,promoter and integration targeting domains to optimize expression.Initially the alpha-factor secretion signal with the standard KEXprotease site is employed to facilitate secretion of heavy and lightchains. The properties of the expression vector may be further refinedusing standard techniques.

An Ig expression vector such as the one described above is introducedinto a host cell of the invention that expresses GnTIII, preferably inthe Golgi apparatus of the host cell. The Ig molecules expressed in sucha host cell comprise N-glycans having bisecting GlcNAcs.

Example 13 Generation of Yeast Strain YSH-1 (Δoch1, α1,2-Mannosidase,GnTI)

The previously reported P. pastoris strain BK64 (Choi et al. (2003) ProcNatl Acad Sci USA. 100(9):5022-7), a triple auxotroph (ADE, ARG, HIS)possessing the OCH1 knock-out and expressing the kringle 3 domain (K3)of human plasminogen, was used as the host strain. BK64 was transformedwith the plasmid pPB103 linearized with the restriction enzyme EcoNI tointroduce the K. lactis UDP-N-acetylglucosamine transporter into thehost cell, thus creating the strain PBP-1. The mouse MnsI was introducedinto this strain by transformation with the plasmid pFB8 linearized withthe restriction enzyme EcoNI, generating strain PBP-2. K3 glycananalysis from proteins isolated from strain PBP-2 demonstrated that theprimary glycoform present was Man₅GlcNAc₂.

PBP-2 was subsequently transformed with the human GnTI plasmid pNA15linearized with the restriction enzyme AatII, generating the strainPBP-3. Analysis of the K3 glycoforms produced in strain PBP-3demonstrated that the hybrid glycan GlcNAcMan₅GlcNAc₂ was thepredominant structure. To recover the URA3 marker from PBP-3, thisstrain was grown in YPD prior to selection on minimal media containing5-Fluoroorotic (5-FOA, BioVectra) and uracil (Boeke et al. (1984) Mol.Gen. Genet. 197:345-346). The recovered Ura-minus strain producingGlcNAcMan₅GlcNAc₂ glycoforms was designated YSH-1 (FIG. 36). TheN-glycan profile from strain YSH-1 is shown in FIG. 25 (top) anddisplays a predominant peak at 1465 m/z corresponding to the mass ofGlcNAcMan₅GlcNAc₂ [d].

Example 14 Generation of Yeast Strain YSH-37 (P. pastoris ExpressingMannosidase II)

YSH-1 (Example 13) was transformed with the D. melanogaster mannosidaseIIΔ74/S. cerevisiae MNN2(s) plasmid (pKD53) linearized with therestriction enzyme ApaI, generating strain YSH-37 (FIG. 36). Analysis ofthe K3 glycan structures produced in strain YSH-37 (FIG. 25 (bottom))demonstrated that the predominant glycoform at 1140 m/z corresponds tothe mass of GlcNAcMan₃GlcNAc₂ [b] and other glycoforms GlcNAcMan₄GlcNAc₂[c] at 1303 m/z and GlcNAcMan₅GlcNAc₂ [d] at 1465 m/z.

Example 15 Generation of Yeast Strain YSH-44

Strain YSH-37 (Example 14) was transformed with a plasmid encoding a ratGnTII/MNN2(s) leader, pTC53, linearized with the restriction enzymeEcoRI. The resulting strain, YSH-44 (FIG. 36), produced a K3 N-glycanhaving a single glycoform at 1356 m/z, corresponding to the mass ofGlcNAc₂Man₃GlcNAc₂ [x], by positive mode MALDI-TOF mass spectrometry(FIG. 29).

Example 16 Construction of Plasmid pJN 348

The plasmid pBLURA-SX (from Jim Cregg) was digested with BamHI and BglIIto release the AOX expression cassette. The BamHI fragment containingthe GAPDH/CYC1 expression cassette from pJN261 (FIG. 4B) (Example 4) wasthen ligated into the pBLURA-SX backbone to create pJN338. The plasmidpJN338 was cut with NotI and PacI and the two oligonucleotides

5′-GGCCGCCTGCAGATTTAAATGAATTCGGCGCGCCTTAAT-3′ (SEQ ID NO:96) and

5′-TAAGGCGCGCC GAATTCATTTAAATCTGCAGGGC-3′ (SEQ ID NO:97) that had beenannealed in vitro, were ligated into the open sites, to create pJN348.

Example 17 Construction of an Integration Plasmid pRCD259

The PpURA3 containing GAPDH expression vector pJN348 was linearized withXhoI and blunted with T4 DNA polymerase and calf intestinal phosphatase(CIP) treated. The HYG resistance marker was digested from pAG32 withBglII and SacI and blunted, then ligated into pJN348 to create pRCD259which can be used as a HYG expression vector that integrates at thePpURA3 locus.

Example 18 Generation of GnTIII Fusion Constructs

Fusion constructs between mammalian GnTIII and yeast targeting sequenceswere made using mouse Mgat3 gene (GenBank accession number L39373,Bhaumik et al., 1995). Three DNA fragments corresponding to N-terminaldeletions Δ32, Δ86, and Δ212 of the mouse GnTIII gene were PCR amplifiedusing Pfu Turbo polymerase (Stratagene) with forward

MG3-B (5′-TCCTGGCGCGCCTTCCCGAGAGAACTGGCCTCCCTC-3′) (SEQ ID NO:98),

MG3-C (5′-CCGAGGCGCGCCACAGAGGAACTGCACCGGGTG-3′) (SEQ ID NO:99),

MG3-D (5′-ACCGAGGCGCGCCATCAACGCCATCAACATCAACCAC-3′) (SEQ ID NO:100),

and reverse

MG3-A (5′-AATTAATTAACCCTAGCCCTCCGCTGTATCCAACTTG-3′) (SEQ ID NO:101)primers. The PCR products were then cloned into pJN 348 vector asAscI-PacI fragments and sequenced. The resulting vectors pVA (GnTIIIΔ32), pVB (GnTIII Δ86), and pVC (GnTIII Δ212) were digested withNotI-AscI enzymes and used for the ligation with yeast leader library(leaders 20-67). These targeting peptides are fused to the catalyticdomains selected from the mouse GnTIII with 32, 86, 212 amino acidN-terminal deletions. For example, the MNN2 targeting peptide from S.cerevisiae (long, medium and short) and GNT1 from K. lactis (short, andmedium) (see Example 11) are shown in Table 11.

TABLE 11 A representative combinatorial library of targeting peptidesequences/catalytic domains exhibiting UDP-N-Acetylglucosaminyltransferase III (GnTIII) activity in P. pastoris YSH-1Targeting peptide S. cerevisiae S. cerevisiae S. cerevisiae K. lactisMNN2(s) MNN2(m) MNN2(I) GNT1(m) Catalytic Domian Mouse GnTIII 50% 30-40%20-30% 0% Δ32 (pVA53) (pVA54) (pVA55) (pVA51) Mouse GnTIII 20-30% 30-40%20-30% 0% Δ86 (pVB53) (pVB54) (pVB55) (pVB51) Mouse GnTIII  0% 0% 0% 0%Δ212 (pVC53) (pVC54) (pVC55) (pVC51)

Example 19 Engineering of P. pastoris to Produce BisectedGlcNAc₂Man₅GlcNAc₂

The P. pastoris strain producing GlcNAcMan₅GlcNAc₂ (PBP-3) (see Example8) was counterselected on 5-FOA, thereby selecting for loss of the URA3+marker and a ura3− phenotype. This strain, designated YSH-1 (FIG. 36),was transformed with the library of N-acetylglucosaminyltransferase III(GnTIII) catalytic domains (vectors pVA, pVB, and pVC) and leaders.Transformants were grown at 30° C. in BMGY to an OD600 of about 10,harvested by centrifugation and transferred to BMMY to induce theproduction of K3 (kringle 3 from human plasminogen) under control of anAOX1 promoter. K3 was purified from the medium by Ni-affinitychromatography utilizing a 96-well format on a Beckman BioMek 2000laboratory robot. The robotic purification is an adaptation of theprotocol provided by Novagen for their HisBind resin (Example 3). TheN-glycans were released by PNGase digestion (Example 3). The N-glycanswere analyzed with a MALDI-TOF MS (Example 3). The GnTIII activities areshown in Table 11. The number of (+)s, as used herein, indicates therelative levels of bisected N-glycan production of % neutral glycans.Targeting peptide sequences were selected from selected from the groupconsisting of: Saccharomyces GLS1, Saccharomyces MNS1, SaccharomycesSEC12, Pichia SEC, Pichia OCH 1, Saccharomyces MNN9, Saccharomyces VAN1, Saccharomyces ANP1, Saccharomyces HOC1, Saccharomyces MNN10,Saccharomyces MNN11, Saccharomyces MNT1, Pichia D2, Pichia D9, PichiaJ3, Saccharomyces KTR1, Saccharomyces KTR2, Kluyveromyces GnTI,Saccharomyces MNN2, Saccharomyces MNN5, Saccharomyces YUR1,Saccharomyces MNN1, and Saccharomyces MNN6. The pVA53 transformantsexhibiting the bisecting GlcNAc (e.g. GlcNAc₂Man₅GlcNAc₂) weredesignated PBP26 (FIG. 36).

Example 20 Engineering of P. pastoris YSH-44 to Produce BisectedGlcNAc₃Man₃GlcNAc₂

For the expression of GnTIII in the strain YSH-44 (FIG. 36), GnTIIIconstructs from vectors pVA53, pVB53, pVA54, and pVB54 were transferredas NotI-PacI fragments into pRCD259 to generate vectors pPB135, pPB137,pPB136, and pPB138. The vectors contain HYG resistance marker and P.pastoris URA3 gene as targeting sequence for genomic integration.Plasmids are linearized with SalI, transformed into strain YSH-44 byelectroporation, selected on medium containing hygromycin and theresulting strains are screened by analysis of the released glycans frompurified K3. Transformants were grown at 24° C. in BMGY to an OD600 ofabout 10, harvested by centrifugation and transferred to BMMY to inducethe production of K3 (kringle 3 from human plasminogen) under control ofan AOX1 promoter. K3 was purified from the medium by Ni-affinitychromatography utilizing a 96-well format on a Beckman BioMek 2000laboratory robot (Example 3). The robotic purification is an adaptationof the protocol provided by Novagen for their HisBind resin (Example 3).The N-glycans were released by PNGase digestion. The N-glycans wereanalyzed with a MALDI-TOF MS (Example 3). The pPB135 transformantsexhibiting the bisecting GlcNAc (e.g. GlcNAc₂Man₅GlcNAc₂) weredesignated YSH-57 (FIG. 36). Table 11 depicts the activity of the mouseGnTIII.

Example 21 Engineering of P. pastoris PBP6-5 to Produce BisectedGlcNAc₃Man₃GlcNAc₂

The P. pastoris PBP6-5 (Example 11) was transformed with the plasmidpPB135 (Table 11) encoding a mouse GnTIII catalytic domain (Δ32) ligatedin frame to a targeting peptide derived from S. cerevisiae MNN2.Transformants were grown at 30° C. in BMGY to an OD600 of about 10,harvested by centrifugation and transferred to BMMY to induce theproduction of K3 (kringle 3 from human plasminogen) under control of anAOX1 promoter. K3 was purified from the medium by Ni-affinitychromatography utilizing a 96-well format on a Beckman BioMek 2000laboratory robot. The robotic purification is an adaptation of theprotocol provided by Novagen for their HisBind resin (Example 3). TheN-glycans were released by PNGase digestion (Example 3). The N-glycanswere analyzed with a MALDI-TOF MS (Example 3). Transformants exhibitingthe bisecting GlcNAc (e.g. GlcNAc₂Man₃GlcNAc₂) were designated PBP-38(FIG. 36). Table 11 depicts the activity of the mouse GnTIII.

Example 22 In Vitro GnTIII Activity Assay Using SubstrateGlcNAcMan₅GlcNAc₂ in Engineered P. pastoris Strain YSH-57

To test any potential ex vivo GnTIII activity in the P. pastoris strain,YSH-57 cell culture supernatants were tested for GnTIII activity. P.pastoris YSH-57 cells were grown at 24° C. in BMGY to an OD600 of about10. Cells were harvested by centrifugation and transferred to BMMY toinduce the production of K3 (kringle 3 from human plasminogen) undercontrol of an AOX1 promoter. After 24 hours of induction, cells wereremoved by centrifugation to yield an essentially clear supernatant. Analiquot of the supernatant was removed for GnTIII assays and theremainder was used for the recovery of secreted soluble K3. K3 waspurified from the medium by Ni-affinity chromatography utilizing a96-well format on a Beckman BioMek 2000 laboratory robot. The roboticpurification is an adaptation of the protocol provided by Novagen fortheir HisBind resin (Example 3). The N-glycans were released by PNGasedigestion (Example 3). The earlier removed aliquot of the supernatantwas further tested for the presence of secreted GnTIII activity.GlcNAcMan₅GlcNAc₂ purified from K3 expressed in PBP-3 strain was addedto: BMMY (A) 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) inBMMY (B); the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C);the supernatant of YSH-57+1 mM UDP-GlcNAc (D). After incubation for 8hours at room temperature, samples were analyzed by amino silica HPLC todetermine the extent of GnTIII activity.

Example 23 In Vitro GnTIII Activity Assay Using SubstrateGlcNAc₂Man₃GlcNAc₂ in Engineered P. pastoris Strain YSH-57

To test any potential ex vivo GnTIII activity in the P. pastoris strainYSH-57 cell culture supernatants were tested for GnTIII activity. P.pastoris YSH-57 cells were grown at 24° C. in BMGY to an OD600 of about10. Cells were harvested by centrifugation and transferred to BMMY toinduce the production of K3 (kringle 3 from human plasminogen) undercontrol of an AOX1 promoter. After 24 hours of induction, cells wereremoved by centrifugation to yield an essentially clear supernatant. Analiquot of the supernatant was removed for GnTIII assays and theremainder was used for the recovery of secreted soluble K3. K3 waspurified from the medium by Ni-affinity chromatography utilizing a96-well format on a Beckman BioMek 2000 laboratory robot. The roboticpurification is an adaptation of the protocol provided by Novagen fortheir HisBind resin (Example 3). The N-glycans were released by PNGasedigestion (Example 3). The earlier removed aliquot of the supernatantwas further tested for the presence of secreted GnTIII activity.GlcNAc₂Man₃GlcNAc₂ purified from K3 expressed in YSH-44 strain was addedto: BMMY (A) 1 mM UDP-GlcNAc (Sigma Chemical Co., St. Louis, Mo.)) inBMMY (B); the supernatant of YSH-44 transformed with pVA53 [YSH-57] (C).After incubation for 8 hours at room temperature, samples were analyzedby amino silica HPLC to determine the extent of GnTIII activity.

1. A yeast host cell that has been genetically engineered to bediminished or depleted in the activity of an initiatingα-1,6-mannosylatransferase and to comprise a nucleic acid sequenceencoding an α-1,2-mannosidase, a nucleic acid sequence encoding aN-acetylglucosaminyltransferase I (GnT I), and a nucleic acid sequenceencoding an N-acetylglucosaminyltransferase III (GnT III).
 2. The yeasthost cell of claim 1, wherein the GnT III has intracellular catalyticactivity.
 3. The yeast host cell of claim 1, further comprising anucleic acid sequence encoding mannosidase II.
 4. The yeast host cell ofclaim 1, wherein the GnT III produces a bisected glycan.
 5. The yeasthost cell of claim 1, wherein the initiating α-1,6-mannosylatransferaseis OCH1.
 6. The yeast host cell of claim 1, wherein the yeast is furtherdiminished or depleted in the activity of Dol-P-Man:Man5GlcNAc2-PP-Dolmannosyltransferase.
 7. The yeast host cell of claim 1, furthercomprising a nucleic acid sequence encoding a UDPN-acetylglucosaminyltransferase (GlcNAc) transporter.
 8. The yeast hostcell of claim 1, wherein the yeast is methylotrophic.
 9. A yeast hostcell that has been genetically engineered to be diminished or depletedin the activity of an initiating α-1,6-mannosylatransferase and tocomprise a nucleic acid sequence encoding an α-1,2-mannosidase, anucleic acid sequence encoding an N-acetylglucosaminyltransferase I (GnTI), a nucleic acid sequence encoding an N-acetylglucosaminyltransferaseII (GnT II), and a nucleic acid sequence encoding anN-acetylglucosaminyltransferase III (GnT III).
 10. The yeast host cellof claim 9, wherein the GnT II and the GnT III have intracellularcatalytic activity.
 11. The yeast host cell of claim 9, furthercomprising a nucleic acid sequence encoding mannosidase II.
 12. Theyeast host cell of claim 9, wherein the GnT III produces a bisectedglycan.
 13. The host cell of claim 9, wherein the host cell is selectedfrom the group consisting of Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,and Candida albicans.
 14. The yeast host cell of claim 9, wherein theinitiating α-1,6-mannosylatransferase is OCH1.
 15. The yeast host cellof claim 9, wherein the yeast is further diminished or depleted in theactivity of Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase.
 16. Theyeast host cell of claim 9, further comprising a nucleic acid sequenceencoding a UDP N-acetylglucosaminyltransferase (GlcNAc) transporter. 17.The yeast host cell of claim 9, wherein the yeast is methylotrophic. 18.A yeast host cell that has been genetically engineered to be diminishedor depleted in the activity of an initiating α-1,6-mannosylatransferaseand to comprise a nucleic acid sequence encoding an α-1,2-mannosidase, anucleic acid sequence encoding a N-acetylglucosaminyltransferase I (GnTI), a nucleic acid sequence encoding an N-acetylglucosaminyltransferaseIII (GnT III), and a nucleic acid sequence encoding a mannosidase II.19. The yeast host cell of claim 18, wherein the initiatingα-1,6-mannosylatransferase is OCH1.
 20. The yeast host cell of claim 18,wherein the yeast is further diminished or depleted in the activity ofDol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase.
 21. The yeast hostcell of claim 18, further comprising a nucleic acid sequence encoding aUDP N-acetylglucosaminyltransferase (GlcNAc) transporter.
 22. The yeasthost cell of claim 18, wherein the yeast is methylotrophic.